Antibodies reactive with VEGF-C, a ligand for the Flt4 receptor tyrosine kinase (VEGFR-3)

ABSTRACT

The invention provides antibodies that are reactive with VEGF-C, a polypeptide ligand for Flt4 receptor tyrosine kinase (VEGFR-3).

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/585,895, filed Jan. 12, 1996, now U.S. Pat. No. 6,245,530,which is a continuation-in-part of U.S. patent application Ser. No.08/510,133, filed Aug. 1, 1995, now U.S. Pat. No. 6,221,839.

FIELD OF THE INVENTION

The present invention generally relates to the field of geneticengineering and more particularly to growth factors for endothelialcells and growth factor genes.

BACKGROUND OF THE INVENTION

Developmental growth, the remodelling and regeneration of adult tissues,as well as solid tumor growth, can only occur when accompanied by bloodvessel formation. Angioblasts and hematopoietic precursor cellsdifferentiate from the mesoderm and form the blood islands of the yolksac and the primary vascular system of the embryo. The development ofblood vessels from these early (in situ) differentiating endothelialcells is termed vasculogenesis. Major embryonic blood vessels arebelieved to arise via vasculogenesis, whereas the formation of the restof the vascular tree is thought to occur as a result of vascularsprouting from pre-existing vessels, a process called angiogenesis,Risau, et al., Devel. Biol., 125:441-450 (1988).

Endothelial cells give rise to several types of functionally andmorphologically distinct vessels. When organs differentiate and begin toperform their specific functions, the phenotypic heterogeneity ofendothelial cells increases. Upon angiogenic stimulation, endothelialcells may re-enter the cell cycle, migrate, withdraw from the cell cycleand subsequently differentiate again to form new vessels that arefunctionally adapted to their tissue environment. Endothelial cellsundergoing angiogenesis degrade the underlying basement membrane andmigrate, forming capillary sprouts that project into the perivascularstroma. Ausprunk, et al., Microvasc. Rev., 14:51-65 (1977). Angiogenesisduring tissue development and regeneration depends on the tightlycontrolled processes of endothelial cell proliferation, migration,differentiation, and survival. Dysfunction of the endothelial cellregulatory system is a key feature of many diseases. Most significantly,tumor growth and metastasis have been shown to be angiogenesisdependent. Folkman, et al., J. Biol. Chem., 267:10931-10934 (1992).

Key signals regulating cell growth and differentiation are mediated bypolypeptide growth factors and their transmembrane receptors, many ofwhich are tyrosine kinases. Autophosphorylated peptides within thetyrosine kinase insert and carboxyl-terminal sequences of activatedreceptors are commonly recognized by kinase substrates involved insignal transduction for the readjustment of gene expression inresponding cells. Several families of receptor tyrosine kinases havebeen characterized. Van der Geer, et al., Ann. Rev. Cell Biol.,10:251-337 (1994). The major growth factors and receptors transducingangiogenic stimuli are schematically shown in FIG. 1.

Fibroblast growth factors are also known to be involved in theregulation of angiogenesis. They have been shown to be mitogenic andchemotactic for cultured endothelial cells. Fibroblast growth factorsalso stimulate the production of proteases, such as collagenases andplasminogen activators, and induce tube formation by endothelial cells.Saksela, et al., Ann. Rev. Cell Biol., 4:93-126 (1988). There are twogeneral classes of fibroblast growth factors, FGF-1 and FGF-2, both ofwhich lack conventional signal peptides. Both types have an affinity forheparin and FGF-2 is bound to heparin sulfate proteoglycans in thesubendothelial extracellular matrix from which it may be released afterinjury. Heparin potentiates the stimulation of endothelial cellproliferation by angiogenic FGFs, both by protecting againstdenaturation and degradation and dimerizing the FGFs. Culturedendothelial cells express the FGF-1 receptor but no significant levelsof other high-affinity fibroblast growth factor receptors.

Among other ligands for receptor tyrosine kinases, the platelet derivedgrowth factor, PDGF-BB, has been shown to be weakly angiogenic in thechick chorioallantoic membrane. Risau, et al., Growth Factors, 7:261-266(1992). Transforming growth factor α (TGFα) is an angiogenic factorsecreted by several tumor cell types and by macrophages. Hepatocytegrowth factor (HGF), the ligand of the c-met proto-oncogene-encodedreceptor, also is strongly angiogenic.

Recent evidence shows that there are endothelial cell specific growthfactors and receptors that may be primarily responsible for thestimulation of endothelial cell growth, differentiation and certaindifferentiated functions. The best studied of these is vascularendothelial growth factor (VEGF), a member of the PDGF family. Vascularendothelial growth factor is a dimeric glycoprotein of disulfide-linked23 kDa subunits. Other reported effects of VEGF include the mobilizationof intracellular calcium, the induction of plasminogen activator andplasminogen activator inhibitor-1 synthesis, stimulation of hexosetransport in endothelial cells, and promotion of monocyte migration invitro. Four VEGF isoforms, encoded by distinct mRNA splice variants,appear to be equally capable of stimulating mitogenesis in endothelialcells. However, each isoform has a different affinity for cell surfaceproteoglycans, which behave as low affinity receptors for VEGF. The 121and 165 amino acid isoforms of VEGF (VEGF121 and VEGF165) are secretedin a soluble form, whereas the isoforms of 189 and 206 amino acidresidues remain cell surface associated and have a strong affinity forheparin.

VEGF was originally purified from several sources on the basis of itsmitogenic activity toward endothelial cells, and also by its ability toinduce microvascular permeability, hence it is also called vascularpermeability factor (VPF). VEGF produces signals through two receptortyrosine kinases, VEGFR-1 (FLT-1) and VEGFR-2 (KDR/Flk-1), which areexpressed specifically on endothelial cells. The VEGF-related placentagrowth factor (PlGF) was recently shown to bind to VEGFR-1 with highaffinity. PlGF was able to enhance the growth factor activity of VEGF,but it did not stimulate endothelial cells on its own. Naturallyoccurring VEGF/PlGF heterodimers were nearly as potent mitogens as VEGFhomodimers for endothelial cells.

The pattern of VEGF expression suggests its involvement in thedevelopment and maintenance of the normal vascular system and in tumorangiogenesis. During murine development, the entire 7.5 day post-coital(p.c.) endoderm expresses VEGF and the ventricular neuroectodermproduces VEGF at the capillary ingrowth stage. See Breier, et al.,Development, 114:521-523 (1992). On day two of quail development, thevascularized area of the yolk sac as well as the whole embryo showexpression of VEGF. In addition, epithelial cells next to fenestratedendothelia in adult mice show persistent VEGF expression, suggesting arole in the maintenance of this specific endothelial phenotype andfunction.

Two high affinity receptors for VEGF have been characterized. These areVEGFR-1/Flt-1 (fms-like tyrosine kinase-1) and VEGFR-2/Kdr/Flk-1 (kinaseinsert domain containing receptor/fetal liver kinase-1). Those receptorsare classified in the PDGF-receptor family, but they have seven ratherthan five immunoglobulin-like loops in their extracellular domain andthey possess a longer kinase insert than normally observed in thisfamily. The expression of VEGF receptors occurs mainly in vascularendothelial cells, although some may be present on monocytes andmelanoma cells. Only endothelial cells have been reported to proliferatein response to VEGF, and endothelial cells from different sources showdifferent responses. Thus, the signals mediated through VEGFR-1 andVEGFR-2 appear to be cell type specific.

The Flt4 receptor tyrosine kinase (VEGFR-3) is closely related instructure to the products of the VEGFR-1 and VEGFR-2 genes. Despite thissimilarity, the mature form of Flt4 differs from the VEGF receptors inthat it is proteolytically cleaved in the extracellular domain into twodisulfide-linked polypeptides. Pajusola et al., Cancer Res.,52:5738-5743 (1992). The 4.5 and 5.8 kb Flt4 mRNAs encode polypeptideswhich differ in their C-termini due to the use of alternative 3′ exons.The VEGFs do not show specific binding to Flt4 or induce itsautophosphorylation.

Expression of Flt4 appears to be more restricted than expression ofVEGFR-1 or VEGFR-2. The expression of Flt4 first becomes detectable byin situ hybridization in the angioblasts of head mesenchyme, thecardinal vein, and extraembryonically in the allantois of 8.5 day p.c.mouse embryos. In 12.5 day p.c. embryos the Flt4 signal is observed indeveloping venous and presumptive lymphatic endothelia, but arterialendothelia appear negative. During later stages of development, Flt4mRNA becomes restricted to developing lymphatic vessels. Only thelymphatic endothelia and some high endothelial venules express Flt4 mRNAin adult human tissues and increased expression occurs in lymphaticsinuses in metastatic lymph nodes and in lymphangioma. These resultssupport the theory of the venous origin of lymphatic vessels.

Five endothelial cell specific receptor tyrosine kinases, Flt-1(VEGFR-1), KDR/Flk-1 (VEGFR-2), Flt4, Tie and Tek/Tie-2 have so far beendescribed, which possess the intrinsic tyrosine kinase activityessential for signal transduction. Targeted mutations inactivatingFlt-1, Flk-1, Tie and Tek in mouse embryos have indicated theiressential and specific roles in vasculogenesis and angiogenesis at themolecular level. VEGFR-1 and VEGFR-2 bind VEGF with high affinity (Kd 16pM and 760 pM, respectively) and VEGFR-1 also binds the related placentagrowth factor (PlGF; Kd about 200 pM), while the ligands for Tie, Tek,and Flt4 have not heretofore been reported.

SUMMARY OF THE INVENTION

The present invention provides a ligand for the Flt4 receptor tyrosinekinase. Thus, the invention provides a purified and isolated polypeptidewhich specifically binds to the Flt4 receptor tyrosine kinase. In apreferred embodiment, the ligand comprises a fragment of the amino acidsequence shown in SEQ ID NO: 33 which specifically binds to the humanFlt4 receptor tyrosine kinase.

The present invention also provides one or more precursors of an Flt4ligand, wherein one such precursor (designated “prepro-VEGF-C”)comprises the complete amino acid sequence (amino acid residues −102 to317) shown in SEQ ID NO: 33. Thus, the invention includes a purified andisolated polypeptide having the amino acid sequence of residues −102 to317 shown in SEQ ID NO: 33.

A putative 102 amino acid leader (prepro) peptide has been identified inthe amino acid sequence shown in SEQ ID NO: 33. Thus, in a relatedaspect, the invention includes a purified and isolated polypeptidehaving the amino acids sequence of residues 1-317 shown in SEQ ID NO:33.

The expressed Flt4 ligand precursor is proteolytically cleaved uponexpression to produce an approximately 23 kD peptide which is the Flt4ligand (herein designated VEGF-C). Thus, in a preferred embodiment ofthe invention, an Flt4 ligand is provided which is the cleavage productof the precursor peptide shown in SEQ ID NO: 33 and which has amolecular weight of approximately 23 kD under reducing conditions. Aputative VEGF-C precursor, having an observed molecular weight of about32 kD, also is considered an aspect of the invention.

From the foregoing, it will be apparent that an aspect of the inventionincludes a fragment of the purified and isolated polypeptide having theamino acid sequence of residues −102 to 317 shown in SEQ ID NO: 33, thefragment being capable of specifically binding to Flt4 receptor tyrosinekinase. In a preferred embodiment, the invention includes a fragmenthaving an apparent molecular weight of approximately 23 kD as assessedby SDS-PAGE under reducing conditions.

In a related aspect, the invention includes a purified and isolatedpolypeptide capable of specifically binding to Flt4 receptor tyrosinekinase, the polypeptide comprising a fragment of the purified andisolated polypeptide having the amino acid sequence of residues −102 to317 shown in SEQ ID NO: 33, the fragment being capable of specificallybinding to Flt4 receptor tyrosine kinase. Similarly, the inventionincludes a polypeptide having an amino acid sequence comprising aportion of SEQ ID NO:2, the portion encoding a fragment capable ofspecifically binding to Flt4.

Evidence suggests that the amino acids essential for retaining Flt4ligand activity are contained within approximately amino acids 1-115 ofSEQ ID NO: 33, and that a carboxy-terminal proteolytic cleavage toproduce a mature, naturally-occurring Flt4 ligand occurs withinapproximately amino acids 115-180 of SEQ ID NO: 33. Accordingly,preferred polypeptides of the invention include polypeptides comprisingamino acids 1-115, 1-116, 1-117, 1-118, 1-119, 1-120, 1-121, 1-122,1-123, 1-124 . . . 1-178, 1-179, and 1-180 of SEQ ID NO: 33, wherein thepolypeptides specifically bind to an Flt4 receptor tyrosine kinase. Apreferred Flt4 ligand comprises approximately amino acids 1-115 of SEQID NO: 33. Another preferred polypeptide of the invention comprisesapproximately amino acids 1-180 of SEQ ID NO: 33.

The present invention also provides a cDNA encoding a novel polypeptide,designated VEGF-C, that is structurally homologous to VEGF. VEGF-C is aligand for the FLT4 receptor tyrosine kinase (VEGFR-3), a receptortyrosine kinase related to VEGFR-1 and VEGFR-2 that does not bind VEGF.VEGFR-3 is expressed in venous and lymphatic endothelia of fetal tissuesand predominantly in lymphatic endothelial of adult tissues. Kaipainenet al., Cancer Res., 54:6571-77 (1994); Kaipainen et al., Proc. Natl.Acad. Sci. USA, 92:3566-70 (1995).

Thus, aspects of the invention include purified and isolated nucleicacids encoding polypeptides and polypeptide fragments of the invention;vectors which comprise nucleic acids of the invention; and host cellstransformed or transfected with nucleic acids or vectors of theinvention. For example, in a preferred embodiment, the inventionincludes a purified and isolated nucleic acid (e.g., a DNA or an RNA)encoding an Flt4 ligand precursor. Due to the degeneracy of the geneticcode, numerous such coding sequences are possible, each having in commonthe coding of the amino acid sequence shown in SEQ ID NO: 33. As setforth above, the invention includes polypeptides which comprise aportion of the amino acid sequence shown in SEQ ID NO: 33 and which bindthe Flt4 receptor tyrosine kinase (herein designated VEGFR-3); theinvention also is intended to include nucleic acids encoding thesepolypeptides. Ligand precursors according to the invention, whenexpressed in an appropriate host cell, produce, via cleavage, a peptidewhich binds specifically to the Flt4 receptor tyrosine kinase (VEGFR-3).The nucleotide sequence shown in SEQ ID NO:32 contains a preferrednucleotide sequence encoding the Flt4 ligand (VEGF-C).

The present invention also provides a cell line which produces an Flt4ligand. In a preferred embodiment, the ligand may be purified andisolated directly from the cell culture medium. Also provided arevectors comprising a DNA encoding the Flt4 ligand, and host cellscomprising the vectors. Preferred vectors of the invention areexpression vectors wherein nucleic acids of the invention areoperatively connected to appropriate promoters and other controlsequences, such that appropriate host cells transformed or transfectedwith the vectors are capable of expressing the Flt4 ligand. A preferredvector of the invention is plasmid pFLT4-L, having ATCC accession no.97231.

The invention further includes a method of making polypeptides of theinvention. In a preferred method, a nucleic acid or vector of theinvention is expressed in a host cell, and a polypeptide of theinvention is purified from the host cell or the host cell's growthmedium.

In a related embodiment, the invention includes a method of making apolypeptide capable of specifically binding to Flt4 receptor tyrosinekinase, comprising the steps of: (a) transforming or transfecting a hostcell with a nucleic acid of the invention; (b) cultivating the host cellto express the nucleic acid; and (c) purifying a polypeptide capable ofspecifically binding to Flt4 receptor tyrosine kinase from the host cellor from the host cell's growth media.

The invention also is intended to include purified and isolatedpolypeptide ligands of Flt4 produced by methods of the invention.

In another aspect, the invention includes an antibody which isspecifically reactive with polypeptides of the invention. Antibodies,both monoclonal and polyclonal, may be made against a ligand of theinvention according to standard techniques in the art. Such antibodiesmay be used in diagnostic applications to monitor angiogenesis,vascularization, lymphatic vessels and their disease states, woundhealing, or certain hematopoietic or leukemia cells, or they may be usedto block or activate the Flt4 receptor.

Ligands according to the invention may be labeled with a detectablelabel and used to identify their corresponding receptors in situ.Labeled Flt4 ligand and anti-Flt4 ligand antibodies may be used asimaging agents in the detection of lymphatic vessels, high endothelialvenules, and Flt4 receptors expressed in histochemical tissue sections.The ligand or antibody may be covalently or non-covalently coupled to asuitable supermagnetic, paramagnetic, electron dense, echogenic, orradioactive agent for imaging. Other, non-radioactive labels, such asbiotin and avidin, may also be used.

The present invention also provides diagnostic and clinical applicationsfor claimed ligands. In a preferred embodiment, Flt4 ligands orprecursors are used to accelerate angiogenesis, e.g., during woundhealing, or to promote the endothelial functions of lymphatic vessels. Autility for VEGF-C is suggested as an inducer of angiogenesis also intissue transplantation, in eye diseases, in the formation of collateralvessels around arterial stenoses and into injured tissues afterinfarction. Ligands may be applied in any suitable manner using anappropriate pharmaceutically-acceptable vehicle, e.g., apharmaceutically-acceptable diluent, adjuvant, or carrier. Ligands alsomay be used to quantify future metastatic risk by assaying biopsymaterial for the presence of active receptors or ligands in a bindingassay or kit using detectably-labeled ligand. An Flt4 ligand accordingto the invention also may be used to promote re-growth or permeabilityof lymphatic vessels in, for example, organ transplant patients. Ligandsaccording to the invention also may be used to treat or preventinflammation, edema, aplasia of the lymphatic vessels, lymphaticobstruction, elephantiasis, and Milroy's disease. Finally, Flt4 ligandsmay be used to stimulate lymphocyte production and maturation, and topromote or inhibit trafficking of leukocytes between tissues andlymphatic vessels or to affect migration in and out of the thymus.

Inhibitors of the Flt4 ligand may be used to control endothelial cellproliferation and lymphangiomas. For example, such inhibitors may beused to arrest metastatic growth or spread, or to control other aspectsof endothelial cell expression and growth. Inhibitors includeantibodies, antisense oligonucleotides, and peptides which block theFlt4 receptor, all of which are intended as aspects of the invention.

Polynucleotides of the invention such as those described above,fragments of those polynucleotides, and variants of thosepolynucleotides with sufficient similarity to the non-coding strand ofthose polynucleotides to hybridize thereto under stringent conditionsall are useful for identifying, purifying, and isolating polynucleotidesencoding other (non-human) mammalian forms of VEGF-C. Thus, suchpolynucleotide fragments and variants are intended as aspects of theinvention. Exemplary stringent hybridization conditions are as follows:hybridization at 42° C. in 5×SSC, 20 mM NaPO₄, pH 6.8, 50% formamide;and washing at 42° C. in 0.2×SSC. Those skilled in the art understandthat it is desirable to vary these conditions empirically based on thelength and the GC nucleotide base content of the sequences to behybridized, and that formulas for determining such variation exist. See,e.g., Sambrook et al., Molecular Cloning: a Laboratory Manual, SecondEdition, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory (1989).

Moreover, purified and isolated polynucleotides encoding other(non-human) mammalian VEGF-C forms also are aspects of the invention, asare the polypeptides encoded thereby, and antibodies that arespecifically immunoreactive with the non-human VEGF-C variants. Thus,the invention includes a purified and isolated mammalian VEGF-Cpolypeptide, and also a purified and isolated polynucleotide encodingsuch a polypeptide.

In one embodiment, the invention includes a purified and isolatedpolypeptide having the amino acid sequence of residues 1 to 415 of SEQID NO: 41, which sequence corresponds to a putative mouse VEGF-Cprecursor. The putative mouse VEGF-C precursor is believed to beprocessed into a mature mouse VEGF-C in a manner analogous to theprocessing of the human prepro-polypeptide. Thus, in a related aspect,the invention includes a purified and isolated polypeptide capable ofspecifically binding to an Flt4 receptor tyrosine kinase (e.g., a humanor mouse Flt-4 receptor tyrosine kinase), the polypeptide comprising afragment of the purified and isolated polypeptide having the amino acidsequence of residues 1 to 415 of SEQ ID NO: 41, the fragment beingcapable of specifically binding to the Flt4 receptor tyrosine kinase.The invention further includes purified and isolated nucleic acidsencoding the foregoing polypeptides, such as a nucleic acid comprisingall or a portion of the sequence shown in SEQ ID NO: 40.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic diagram showing major endothelial cell receptortyrosine kinases and growth factors involved in vasculogenesis andangiogenesis.

FIGS. 2A and 2B schematically depicts the construction of the pLTRFlt4lexpression vector.

FIG. 3 schematically depicts the construction of the baculovirus vectorencoding a secreted soluble Flt4 extracellular domain (Flt4EC).

FIG. 4 shows results of stimulation of Flt4 autophosphorylation byconditioned medium from PC-3 cell cultures.

FIGS. 5A, 5B, and 5C show that the major tyrosyl phosphorylatedpolypeptide of Flt4-transfected cells stimulated with PC-3 conditionedmedium is the 125 kD Flt4 polypeptide (VEGFR-3), and also that the Flt4stimulating activity is not adsorbed to heparin-sepharose.

FIG. 6 shows Western immunoblotting analysis of the Flt4 ligand activityisolated from PC-3 conditioned medium.

FIG. 7 shows results of gel electrophoresis of chromatographic fractionsfrom the affinity purification of Flt4 ligand (VEGF-C) isolated fromPC-3 conditioned medium.

FIG. 8 shows results of Western analysis of Flt4 autophosphorylationinduced by either the Flt4 ligand (VEGF-C), VEGF, or PlGF.

FIG. 9A schematically depicts the cloning and analysis of the Flt4ligand, VEGF-C. The VEGF homologous region (dark shaded box) and aminoand carboxyl terminal propeptides (light shaded and unshaded boxes,respectively) as well as putative signal sequence (ss) are depictedbetween 5′ and 3′ untranslated (ut) nucleic acid regions. The cleavagesites for the signal sequence and the amino and carboxyl terminalpropeptides are indicated with triangles.

FIGS. 9B-9D show the nucleotide and deduced amino acid sequence of aFlt4 ligand cDNA (without adaptor and poly-A sequences). The cleavagesite for the putative amino terminal prepro leader sequence is indicatedwith a shaded triangle.

FIGS. 10A-10C show a comparison of the deduced amino acid sequences ofPDGF-A, -B, PlGF-1, VEGF-B167, four VEGF isoforms, and Flt4 ligand(VEGF-C).

FIG. 11 shows the stimulation of autophosphorylation of the Flt4receptor by conditioned medium from cells transfected with the pREP7expression vector containing the VEGF-C-encoding cDNA insert of plasmidFLT4-L.

FIG. 12 shows Northern blotting analysis of the genes encoding VEGF,VEGF-B, AND VEGF-C (indicated by “FLT4-L”) in two human tumor cell linesand in brain tissue.

FIG. 13A is an autoradiograph showing recombinant VEGF-C isolatedfollowing a pulse-chase experiment and electrophoresed via SDS-PAGEunder reducing conditions.

FIG. 13B is a photograph of polyacrylamide gel showing that recombinantVEGF-C forms are disulfide-linked in nonreducing conditions.

FIG. 14A and 14B depict Western blots showing that VEGF-C stimulatesautophosphorylation of VEGFR-2 (KDR) but has no effect on PDGFR-βphosphorylation.

FIG. 15A and FIGS. 15B-15E show that VEGF-C stimulates endothelial cellmigration in a three-dimensional collagen gel assay.

FIG. 16A shows the expression of VEGF-C mRNA in human adult tissues.

FIG. 16B shows the expression of VEGF, VEGF-B, and VEGF-C in selectedhuman fetal tissues.

FIG. 17 schematically depicts the chromosomal localization of the VEGF-Cgene.

FIG. 18 is a Northern blot hybridization study showing the effects ofhypoxia on the mRNA expression of VEGF (VEGF-A), VEGF-B and VEGF-C.

FIGS. 19A and 19B depict autoradiograms from a pulse-chaseimmunoprecipitation experiment wherein cells transfected with a VEGF-Cexpression vector (VEGF-C) and mock transfected cells (M) werepulse-labeled with radioactive amino acids and chased for varyinglengths of time.

DETAILED DESCRIPTION OF THE INVENTION

Described herein is the isolation of a novel vascular endothelial growthfactor and the cloning of a DNA encoding this novel growth factor from acDNA library prepared from the human prostatic adenocarcinoma cell linePC-3. The isolated cDNA encodes a protein which is proteolyticallyprocessed and secreted to cell culture medium. The secreted protein,designated VEGF-C, binds to the extracellular domain of Flt4 and inducestyrosine autophosphorylation of Flt4 and VEGFR-2. VEGF-C also stimulatesthe migration of endothelial cells in collagen gel.

The present invention also is directed to novel growth factorpolypeptides which are ligands for the Flt4 receptor tyrosine kinase(VEGFR-3). Ligands of the invention are members of a family ofplatelet-derived growth factors/vascular endothelial growth factorswhich promote mitosis and proliferation of vascular endothelial cellsand/or mesodermal cells. As described in greater detail in Examples 4and 5, ligands recognizing the Flt4 receptor tyrosine kinase werepurified from a PC-3 prostatic adenocarcinoma cell line (ATCC CRL1435).When applied to a population of cells expressing the Flt4 receptor,ligands of the invention stimulate autophosphorylation, resulting inreceptor activation.

A ligand according to the invention may be expressed as a largerprecursor which is cleaved to produce the ligand. A coexpressed regionin some cases results from alternative splicing of RNA of the ligandgene. Such a co-expressed region may be a function of the particularexpression system used to obtain the ligand. The skilled artisanunderstands that in recombinant production of proteins, additionalsequence may be expressed along with a functional peptide depending uponthe particular recombinant construct used to express the protein, andsubsequently removed to obtain the desired ligand. In some cases therecombinant ligand can be made lacking certain residues of theendogenous/natural ligand. Moreover, it is well-known in thatconservative replacements may be made in a protein which do not alterthe function of the protein. Accordingly, it is anticipated that suchalterations are within the scope of the claims. Moreover, it isanticipated that one or more VEGF-C precursors (the largest putativenative VEGF-C precursor having the complete amino acid sequence fromresidue −102 to residue 317 of SEQ ID NO: 33) is capable of stimulatingthe Flt4 ligand without any further processing, in a manner similar tothat in which VEGF stimulates its receptor in its unprocessed form afterthe secretion and concomitant release of the signal sequence.

Results reported herein show that Flt4 (VEGFR-3) transmits signals forthe VEGF-C novel growth factor. This conclusion is based on the specificbinding of VEGF-C to recombinant Flt4EC (Flt4 extracellular domain)protein and the induction of VEGFR-3 autophosphorylation by medium fromVEGF-C transfected cells. In contrast, neither VEGF nor PlGF showedspecific binding to VEGFR-3 or induced its autophosphorylation.

As set forth in greater detail below, the putative prepro-VEGF-C has adeduced molecular mass of 46,883; a putative prepro-VEGF-C processingintermediate has an observed molecular weight of about 32 kD; and matureVEGF-C isolated form conditioned media has a molecular weight of about23 kD as assessed by SDS-PAGE under reducing conditions. A major part ofthe difference in the observed molecular mass of the purified andrecombinant VEGF-C and the deduced molecular mass of the prepro-VEGF-Cencoded by the VEGF-C open reading frame (ORF) is attributable toproteolytic removal of sequences at the amino-terminal andcarboxyl-terminal regions of the prepro-VEGF-C polypeptide. However,proteolytic cleavage of the putative 102 amino acid prepro-leadersequence is not believed to account for the entire difference betweenthe deduced molecular mass of 46,883 and the observed mass of about 23kD, because the deduced molecular weight of a polypeptide consisting ofamino acids 1-317 of SEQ ID NO:33 is 35,881 kD. It is believed that aportion of the observed difference in molecular weights is attributableto proteolytic removal of amino acid residues in the carboxyl terminalregion of the VEGF-C precursor. By extrapolation from studies of thestructure of PDGF (Heldin, et al., Growth Factors, 8:245-52 (1993)), onecan speculate that the region critical for receptor binding andactivation by VEGF-C is contained within the amino-terminal first 180 orso amino acid residues the secreted of VEGF-C protein lacking theputative prepro leader sequence. In fact, the region critical forreceptor binding and activation by VEGF-C is believed to be containedwithin the first approximately 120 amino acid residues of the secretedVEGF-C protein lacking the prepro leader sequence. Thus, the 23 kDpolypeptide binding VEGFR-3 is likely to represent the VEGF-homologousdomain. After biosynthesis, the nascent VEGF-C polypeptide may beglycosylated at three putative N-linked glycosylation sites identifiedin the deduced VEGF-C amino acid sequence. Polypeptides containingmodifications, such as N-linked glycosylations, are intended as aspectsof the invention.

The carboxyl terminal amino acid sequences, which increase the length ofthe VEGF-C polypeptide in comparison with other ligands of this family,show a pattern of spacing of cysteine residues reminiscent of theBalbiani ring 3 protein (BR3P) sequence (Dignam and Case, Gene,88:133-40 (1990); Paulsson, et al., J. Mol. Biol., 211:331-49 (1990)).This novel C-terminal silk protein-like structural motif of VEGF-C mayfold into an independent domain, which, on the basis of theconsiderations above, is at least partially cleaved off afterbiosynthesis. Interestingly, at least one cysteine motif of the BR3Ptype is also found in the carboxyl terminus of VEGF. In our experimentsboth the putative precursor and cleaved ligand were detected in the cellculture media, although processing was apparently cell-associated on thebasis of the pulse-chase experiments. The determination of theamino-terminal and carboxy-terminal sequences of VEGF-C isolates willallow the identification of the proteolytic processing sites. Thegeneration of antibodies against different parts of the pro-VEGF-Cmolecule will allow the exact determination of the precursor-productrelationship and ratio, their cellular distribution, and the kinetics ofprocessing and secretion.

VEGF-C has a conserved pattern of eight cysteine residues, which mayparticipate in the formation of intra- and interchain disulfide bonds,creating an antiparallel dimeric biologically active molecule, similarto PDGF. Mutational analysis of the cysteine residues involved in theinterchain disulfide bridges has shown that, in contrast to PDGF, VEGFdimers need to be held together by these covalent interactions in orderto maintain biological activity. Disulfide linking of the VEGF-Cpolypeptide chain was evident in the analysis of VEGF-C in nonreducingconditions.

VEGFR-3, which distinguishes between VEGF and VEGF-C, is closely relatedin structure to VEGFR-1 and VEGFR-2. Finnerty, et al, Oncogene,8:2293-98 (1993); Galland, et al., Oncogene, 8:1233-40 (1993); Pajusola,et al., Cancer Res., 52:5738-43 (1992). However, the mature form ofVEGFR-3 differs from the two other VEGFRs in that it is proteolyticallycleaved in the extracellular domain into two disulfide-linkedpolypeptides. Pajusola, et al., Oncogene, 9:3545-55 (1994). Anotherdifference is that the 4.5 and 5.8 kb VEGFR-3 mRNAs encode polypeptidesdiffering in their C-termini and apparently in their signallingproperties due to the use of alternative 3′ exons. Borg et al.,Oncogene, 10:973-84 (1995); Pajusola et al., Oncogene, 8:2931-37 (1993).

Besides VEGFR-3, VEGFR-2 tyrosine kinase also is activated in responseto VEGF-C. VEGFR-2 mediated signals cause striking changes in themorphology, actin reorganization and membrane ruffling of porcine aorticendothelial cells overexpressing this receptor. In these cells, VEGFR-2also mediated ligand-induced chemotaxis and mitogenicity. Waltenbergeret al., J. Biol. Chem, 269:26988-95 (1994). Similarly, the receptorchimera CSF-1R/VEGFR-3 was mitogenic when ectopically expressed in NIH3T3-fibroblastic cells, but not in porcine aortic endothelial cells(Pajusola et al., 1994). Consistent with such results, the bovinecapillary endothelial (BCE) cells, which express VEGFR-2 mRNA but verylittle or no VEGFR-1 or VEGFR-3 mRNAs, showed enhanced migration whenstimulated with VEGF-C. Light microscopy of the BCE cell cultures incollagen gel also suggested that VEGF-C stimulated the proliferation ofthese cells. The already existing data thus indicate that the VEGFligands and receptors show a great specificity in their signalling,which may be cell type dependent.

The expression pattern of the VEGFR-3 (Kaipainen et al., Proc. Natl.Acad. Sci. USA, 92:3566-70 (1995)) suggests that VEGF-C may function inthe formation of the venous and lymphatic vascular systems duringembryogenesis. Constitutive expression of VEGF-C in adult tissues shownherein further suggests that this gene product also is involved in themaintenance of the differentiated functions of the lymphatic endotheliumwhere VEGFR-3 is expressed (Kaipainen et al., 1995). Lymphaticcapillaries do not have well formed basal laminae and an interestingpossibility remains that the silk-like BR3P motif is involved inproducing a supramolecular structure which could regulate theavailability of VEGF-C in tissues. However, as shown here, VEGF-C alsoactivates VEGFR-2, which is abundant in proliferating endothelial cellsof vascular sprouts and branching vessels of embryonic tissues, butdecreased in adult tissues. Millauer et al., Nature, 367:576-78 (1993).These data have suggested that VEGFR-2 is a major regulator ofvasculogenesis and angiogenesis. VEGF-C may thus have a unique effect inlymphatic endothelium and a more redundant function shared with VEGF inangiogenesis and possibly permeability regulation of several types ofendothelia. Because VEGF-C stimulates VEGFR-2 and promotes endothelialmigration, a utility for VEGF-C is suggested as an inducer ofangiogenesis of blood and lymphatic vessels in wound healing, tissuetransplantation, in eye diseases, in the formation of collateral vesselsaround arterial stenoses and into injured tissues after infarction.

Taken together, these results show an increased complexity of signallingin the vascular endothelium. They reinforce the concept that when organsdifferentiate and begin to perform their specific functions, thephenotypic heterogeneity of endothelial cells increases in several typesof functionally and morphologically distinct vessels. However, uponsuitable angiogenic stimuli, endothelial cells can re-enter the cellcycle, migrate, withdraw from the cell cycle and subsequentlydifferentiate again to form new vessels that are functionally adapted totheir tissue environment. This process of angiogenesis concurrent withtissue development and regeneration depends on the tightly controlledbalance between positive and negative signals for endothelial cellproliferation, migration, differentiation and survival.Previously-identified growth factors promoting angiogenesis include thefibroblast growth factors, hepatocyte growth factor/scatter factor, PDGFand TGF-α. (See, e.g., Folkman, Nature Med. 1:27-31 (1995); Friesel andMaciag, FASEB J. 9:919-25 (1995); Mustonen and Alitalo, J. Cell Biol.,129:895-98 (1995). However, VEGF has been the only growth factorrelatively specific for endothelial cells. The newly identified factorsVEGF-B and VEGF-C thus increase our understanding of the complexity ofthe specific and redundant positive signals for endothelial cellsinvolved in vasculogenesis, angiogenesis, permeability and perhaps alsoother endothelial functions.

Also described herein is the localization of the VEGF-C genes in humanchromosomes by analysis of somatic cell hybrids and fluorescence in situhybridization (FISH). Southern blotting and polymerase chain reactionanalysis of somatic cell hybrids and fluorescence in situ hybridizationof metaphase chromosomes was used to assess the chromosomal localizationof the VEGF-C gene. The VEGF-C gene was located on chromosome 4q34,close to the human aspartylglucosaminidase gene previously mapped to4q34-35. The VEGF-C locus in 4q34 is a candidate target for mutationsleading to vascular malformations or cardiovascular diseases. Expressionstudies by Northern blotting and hybridization show abundant VEGF-Cexpression in heart and skeletal muscle; other tissues, such as lung andkidney, also express this gene. Whereas PlGF is predominantly expressedin the placenta, the expression patterns of the VEGFs overlap in manytissues, which suggests that they may form heterodimers and interact toexert their physiological functions.

Targeted mutagenesis leading to inactivation of the VEGF receptor lociin the mouse genome has shown that VEGFR-1 is necessary for the properorganization of endothelial cells forming the vascular endothelium,while VEGFR-2 is necessary for the generation of both endothelial andhematopoietic cells. This suggests that the four genes of the VEGFfamily can be targets for mutations leading to vascular malformations orcardiovascular diseases.

The following Examples illustrate preferred embodiments of theinvention, wherein the isolation, characterization, and function of Flt4ligands and ligand-encoding nucleic acids according to the invention areshown.

EXAMPLE 1 Production of pLTRFlt4l Expression Vector

Construction of the LTR-Flt4l vector is schematically shown in FIGS. 2Aand 2B. The full-length Flt4s cDNA (Genbank Accession No. X68203, SEQ IDNO: 36) was assembled by first subcloning the S2.5 fragment, reported inPajusola et al., Cancer Res. 52:5738-5743 (1992), incorporated byreference herein, containing base pairs 56-2534 of the Flt4s into theEcoRI site of the pSP73 vector (Promega, Madison, Wis.).

Since cDNA libraries used for screening of Flt4 cDNAs did not containits most 5′ protein-coding sequences, inverse PCR was used for theamplification of the 5′ end of Flt4 corresponding to the first 12 aminoacid residues (MQRGAALCLRLW). Poly(A)⁺ RNA was isolated from human HELerythroleukemia cells and double-stranded cDNA copy was synthesizedusing the Amersham cDNA Synthesis System Plus kit and a gene specificprimer: 5′-TGTCCTCGCTGTCCTTGTCT-3′ (SEQ ID NO: 1), which was located 195bp downstream of the 5′ end of clone S2.5. Double stranded cDNA wastreated with T4 DNA polymerase to blunt the ends and cDNA was purifiedwith Centricon 100 filters (Amicon Inc., Beverly, Mass.).Circularization of the blunt-ended cDNA was performed in a total volumeof 150 microliters. The reaction mixture contained ligation buffer, 5%PEG-8000, 1 mM DTT and 8U of T4 DNA ligase (New England Biolabs).Ligation was carried out at 16° C. for 16 hours. Fifteen microliters ofthis reaction mix were used in a standard 100 microliters PCR reactioncontaining 100 ng of specific primers including SacI and PstIrestriction sites, present in this segment of the Flt4 cDNA, and 1 unitof Taq DNA polymerase (Perkin Elmer Cetus). Two rounds of PCR wereperformed using 33 cycles (denaturation at 95° C. for 1 minute,annealing at 55° C. for 2 minutes and elongation at 72° C. for 4minutes). The PCR mixture was treated sequentially with the SacI andPstI restriction enzymes, and after purification with MagicPCR Preps(Promega), DNA fragments were subcloned into the pGEM3Zf(+) vector forsequencing. The sequence obtained corresponds to the 5′ end of the Flt4scDNA clone deposited in the Genbank Database as Accession No. X68203.

The sequence encoding the first 12 amino acid residues was added to theexpression construct by ligating an SphI digested PCR fragment amplifiedusing reverse transcription-PCR of poly(A)⁺ RNA isolated from the HELcells using the oligonucleotides 5′-ACATGCATGC CACCATGCAG CGGGGCGCCGCGCTGTGCCT GCGACTGTGG CTCTGCCTGG GACTCCTGGA-3′ (SEQ ID NO: 2)(forwardprimer, SphI site underlined, the translational start codon marked inbold follows an optimized Kozak consensus sequence Kozak, Nucl. AcidsRes. 15:8125-8148, 1987) and 5′-ACATGCATGC CCCGCCGGT CATCC-3′ (SEQ IDNO: 3) (reverse primer, SphI site underlined) to the 5′ end of the S2.5fragment, thus replacing unique SphI fragment of the S2.5 plasmid. Theresulting vector was digested with EcoRI and ClaI and ligated to a 138bp PCR fragment amplified from the 0.6 kb EcoRI fragment (base pairs3789 to 4416 in the Genbank X68203 sequence) which encodes the 3′ end ofFlt4s shown in FIG. 1 of Pajusola et al., Cancer Res. 52:5738-5743,1992, using the oligonucleotides 5′-CGGAATTCCC CATGACCCCA AC-3′ (SEQ IDNO: 4) (forward, EcoRI site underlined) and 5′-CCATCGATGG ATCCTACCTGAAGCCGCTTT CTT-3′ (SEQ ID NO: 5) (reverse, ClaI site underlined). Thecoding domain was completed by ligation of the 1.2 kb EcoRI fragment(base pairs 2535-3789 of sequence X68203) into the above construct. Thecomplete cDNA was subcloned as a HindIII-ClaI(blunted) fragment (thisClaI site was also included in the 3′ primer used to construct the 3′end of the coding sequence) to the pLTRpoly expression vector reportedin Mäkelä et al, Gene, 118:293-294 (1992) (Genbank accession numberX60280, SEQ ID NO: 37), incorporated by reference herein, using itsHindIII-Acc I(blunted) restriction sites.

The long form of Flt4 was produced by replacing the 3′-end of the shortform as follows: The 3′ region of the Flt41 cDNA was PCR-amplified usinga gene specific and a pGEM 3Z vector specific (SP6 promoter)oligonucleotide 5′-ATTTAGGTGACACTATA-3′ (SEQ ID NO: 6) as reverse andforward primers, respectively, and an Flt4l cDNA clone containing a 495bp EcoRI fragment extending downstream of the EcoRI site at nucleotide3789 of the Genbank X68203 sequence (the sequence downstream of thisEcoRI site is deposited as the Flt4 long form 3′ sequence having Genbankaccession number S66407 (SEQ ID NO: 38)). The gene specificoligonucleotide contained a BamHI restriction site located right afterthe end of the coding region. The sequence of that (reverse primer)oligonucleotide was 5′-CCATCGATGGATCCCGATGCTGCTTAGTAGCTGT-3′ (SEQ ID NO:7) (BamHI site is underlined). The PCR product was digested with EcoRIand BamHI and transferred in frame to LTRFlt4s vector fragment fromwhich the coding sequences downstream of the EcoRI site at base pair2535 (see sequence X68203) had been removed by EcoRI-BamHI digestion.Again, the coding domain was completed by ligation of the 1.2 kb EcoRIfragment (base pairs 2535-3789 of sequence X68203) back into theresulting construct.

EXAMPLE 2 Production and Analysis of Flt41 Transfected Cells

NIH 3T3 cells (60% confluent) were co-transfected with 5 micrograms ofthe pLTRFlt4l construct and 0.25 micrograms of the pSV2neo vector (ATCC)containing the neomycin phosphotransferase gene, using the DOTAPliposome-based transfection reagents (Boehringer Mannheim, Mannheim,Germany). One day after the transfection the cells were transferred intoselection media containing 0.5 mg/ml geneticin (GIBCO, Grand Island,N.Y.). Colonies of geneticin-resistant cells were isolated and analyzedfor expression of the Flt4 proteins. Cells were lysed in boiling lysisbuffer containing 3.3% SDS (sodium dodecyl sulphate), 125 mM Tris, pH6.8. Protein concentrations of the samples were measured by the BCAmethod (Pierce, Rockford, IL). About 50 micrograms of protein of eachlysate were analyzed for the presence of Flt4 by 6% SDS-polyacrylamidegel electrophoresis (SDS-PAGE) and immunoblotting using antisera againstthe carboxyl terminus of Flt4 and the ECL method (Amersham).

For production of anti-Flt4 antiserum the Flt4 cDNA fragment encodingthe 40 carboxy-terminal amino acid residues of the short form:NH2-PMTPTTYKG SVDNQTDSGM VLASEEFEQI ESRHRQESGFR-COOH (SEQ ID NO: 8) wascloned as a 657 bp EcoRI-fragment into the pGEX-1λT bacterial expressionvector (Pharmacia) in frame with the glutathione-S-transferase codingregion. The resultant GST-Flt4S fusion protein was produced in E. coliand purified by affinity chromatography using a glutathione-Sepharose 4Bcolumn. The purified protein was lyophilized, dissolved in phosphatebuffered saline (PBS), mixed with Freund's adjuvant and used forimmunization of rabbits at biweekly intervals using methods standard inthe art (Harlow and Lane, Antibodies, A Laboratory Manual, Cold SpringHarbor Laboratory Press, 1988). Antisera were used after the fourthbooster immunization for immunoprecipitation of Flt4 from thetransfected cells and cell clones expressing Flt4 were used for ligandstimulation analysis.

EXAMPLE 3 Construction of a Flt4 EC Baculovirus Vector and Expressionand Purification of its Product

The construction of an Flt4 extracellular domain (EC) baculovirus vectoris schematically depicted in FIG. 3. The Flt4-encoding cDNA has beenprepared in both a long form and a short form, each being incorporatedin a vector under control of the Moloney murine leukemia virus LTRpromoter. The nucleotide sequence of the short form of the Flt4 receptoris available on the Genbank database as Accession No. X68203 and thespecific 3′ segment of the long form cDNA is available as Accession No.S66407.

The ends of a cDNA segment encoding Flt4 extracellular domain (EC) weremodified as follows: The 3′ end of Flt4 cDNA sequence (Genbank AccessionNumber X68203) which encodes the extracellular domain was amplifiedusing primer 1116 5′-CTGGAGTCGACTTGGCGGACT-3′ (SEQ ID NO: 9, SalI siteunderlined) and primer 13155′-CGCGGATCCCTAGTGATGGTGATGGTGATGTCTACCTTCGATCATGCT GCCCTTAT CCTC-3′(SEQ ID NO: 10, BamHI site underlined). The sequence complementary tothat of primer 1315 continues after the Flt4 reading frame and encodes 6histidine residues for binding to a Ni-NTA column (Qiagen, Hilden,Germany) followed by a stop codon, and an added Bam HI site. Theamplified fragment was digested with SalI and BamHI and used to replacea unique SalI-BamHI fragment in the LTRFlt4 vector shown in FIG. 3. TheSalI-BamHI fragment that was replaced encodes the Flt4 transmembrane andcytoplasmic domains.

The 5′ end without the Flt4 signal sequence encoding region wasamplified by PCR using the primer 13355′-CCCAAGCTTGGATCCAAGTGGCTACTCCATGACC-3′ (SEQ ID NO: 11) (the primercontains added HindIII (AAGCTT) and BamHI (GGATCC, residues 10-15 of SEQID NO: 11) restriction sites, which are underlined) and primer 13325′-GTTGCCTGTGATGTGCACCA-3′ (SEQ ID NO: 12). The amplified fragment wasdigested with HindIII and SphI (the HindIII site (AAGCTT, residues of10-15 of SEQ ID NO: 11) is underlined in primer 1335 and the SphI siteis within the amplified region of the Flt4l cDNA). The resultantHindIII-SphI fragment was used to replace a HindIII-SphI fragment in themodified LTRFlt4l vector described immediately above (the HindIII siteis in the 5′ junction of the Flt4 insert with the pLTRpoly portion ofthe vector, the SphI site is in Flt4 cDNA). The resultant Flt4EC insertwas then ligated as a BamHI fragment into the BamHI site in the pVTBacplasmid as disclosed in Tessier et al., Gene 98: 177-183 (1991),incorporated by reference herein. The orientation was confirmed to becorrect by partial sequencing so that the open reading frame of thesignal sequence-encoding portion of the vector continued in frame withthe Flt4 sequence. That construct was transfected together with thebaculovirus genomic DNA into SF-9 cells by lipofection. Recombinantvirus was purified, amplified and used for infection of High-Five cells(Invitrogen, San Diego, Calif.) using methods standard in the art. TheFlt4 extracellular domain (Flt4EC) was purified from the culture mediumof the infected High-Five cells using Ni-NTA affinity chromatographyaccording to manufacturer's instructions (Qiagen) for binding andelution of the 6xHis tag encoded in the COOH-terminus of the recombinantFlt4 extracellular domain.

EXAMPLE 4 Isolation of Flt4 Ligand from Conditioned Media

An Flt4 ligand according to the invention was isolated from conditionedmedia from PC-3 prostatic adenocarcinoma cell line CRL1435 from theAmerican Type Culture Collection and cultured as instructed by thesupplier in Ham's F-12 Nutrient mixture (GIBCO) containing 7% fetal calfserum. In order to prepare the conditioned media, confluent PC-3 cellswere cultured for 7 days in Ham's F-12 Nutrient mixture (GIBCO) in theabsence of fetal bovine serum. Medium was then cleared by centrifugationat 10,000 g for 20 minutes. The medium was then screened to determineits ability to induce tyrosine phosphorylation of Flt4 by exposure toNIH 3T3 cells which had been transfected with Flt4-encoding cDNA usingthe pLTRFlt4l vector. For receptor stimulation experiments, subconfluentNIH 3T3 cells were starved overnight in serum-free DMEM medium (GIBCO)containing 0.2% BSA. The cells were stimulated with the conditionedmedia for 5 minutes, washed twice with cold PBS containing 100micromolar vanadate and lysed in RIPA buffer (10 mM Tris pH 7.5, 50 mMNaCl, 0.5% sodium deoxycholate, 0.5% Nonidet P40 (BDH, Poole, England),0.1% SDS, 0.1 U/ml Aprotinin (Boehringer Mannheim), 1 mM vanadate) forreceptor immunoprecipitation analysis. The lysates were centrifuged for20 minutes at 15,000×g. The supernatants were incubated for 2 hours onice with 3 microliters of the antiserum against the Flt4 C-terminusdescribed in Example 2 and also in Pajusola, et al. Oncogene 8:2931-2937(1993), incorporated by reference herein.

After a 2 hour incubation in the presence of anti-Flt4 antiserum,protein A-Sepharose (Pharmacia) was added and incubation was continuedfor 45 minutes with rotation. The immunoprecipitates were washed threetimes with the immunoprecipitation buffer and twice with 10 mM Tris, pH7.5, before analysis in SDS-PAGE. Polypeptides were transferred tonitrocellulose and analyzed by Western blotting using Flt4- orphosphotyrosine-specific antisera and the ECL method (AmershamInternational, Buckinghamshire, England). Anti-phosphotyrosinemonoclonal antibodies (anti-PTyr; PY20) were purchased from TransductionLaboratories (Lexington, Ky.). In some cases, the filters were restainedwith a second antibody after stripping. The stripping of the filters wasdone for 30 minutes at 50° C. in 100 mM 2-mercaptoethanol, 2% SDS, 62.5mM Tris-HCl pH 6.7 with occasional agitation.

As shown in FIG. 4, the PC-3 cell conditioned medium stimulated tyrosinephosphorylation of a 125 kD polypeptide when Flt4-expressing NIH 3T3cells were treated with the indicated preparations of media, lysed, andthe lysates were immunoprecipitated with anti-Flt4 antiserum followed bySDS-PAGE, Western blotting, and staining using anti-PTyr antibodies. Theresulting band was weakly phosphorylated upon stimulation withunconcentrated PC-3 conditioned medium (lane 2). The 125 kD bandcomigrated with the tyrosine phosphorylated, processed form of themature Flt4 from pervanadate-treated cells (compare lanes 2 and 7 ofFIG. 4, see also FIG. 5A). Comigration was confirmed upon restainingwith anti-Flt4 antibodies as is also shown in FIG. 5A (panel on theright). In order to show that the 125 kD polypeptide is not anon-specific component of the conditioned medium reactive withanti-phosphotyrosine antibodies, 15 microliters of conditioned mediumwere separated by SDS-PAGE, blotted on nitrocellulose and the blot wasstained with anti-PTyr antibodies. No signal was obtained (FIG. 5B).Also, unconditioned medium failed to stimulate Flt4 phosphorylation, asshown in FIG. 4, lane 1.

FIG. 5C shows a comparison of the effects of PC-3 CM stimulation (+) onuntransfected (lanes 4 and 5), FGFR-4-transfected (lanes 8 and 9) andFlt4-transfected NIH 3T3 cells (lanes 1-3, 6 and 7). These resultsindicate that neither untransfected NIH 3T3 cells nor NIH 3T3 cellstransfected with FGF receptor 4 showed tyrosine phosphorylation of p120upon stimulation with the conditioned medium from PC-3 cells. Analysisof stimulation by PC-3 CM pretreated with Heparin Sepharose CL-6B(Pharmacia) for 2 hours at room temperature (lane 3) shows that the Flt4ligand does not bind to heparin.

As shown in FIG. 4, lane 3, stimulating activity was considerablyincreased when the PC-3 conditioned medium was concentrated four-foldusing a Centricon-10 concentrator (Amicon). FIG. 4, lane 4, shows thatpretreatment of the concentrated PC-3 conditioned medium with 50microliters of the Flt4 extracellular domain coupled to CNBr-activatedsepharose CL-4B (Pharmacia; about 1mg of Flt4EC domain/ml sepharoseresin) completely abolished Flt4 tyrosine phosphorylation. Similarpretreatment of the conditioned medium with unsubstituted sepharoseCL-4B did not affect stimulatory activity, as shown in FIG. 4, lane 5.Also, the flow through obtained after concentration, which containedproteins of less than 10,000 molecular weight, did not stimulate Flt4phosphorylation, as shown in FIG. 4, lane 6.

In another experiment, a comparison of Flt4 autophosphorylation intransformed NIH 3T3 cells expressing LTRFlt4l was conducted, usingunconditioned medium, medium from PC-3 cells expressing the Flt4 ligand,or unconditioned medium containing either 50 ng/ml of VEGF165 or 50ng/ml of PlGF-1. The cells were lysed, immunoprecipitated usinganti-Flt4 antiserum and analyzed by Western blotting usinganti-phosphotyrosine antibodies. As shown in FIG. 8, only the PC-3conditioned medium expressing the Flt4 ligand (lane Flt-4L) stimulatedFlt4 autophosphorylation.

The foregoing data show that PC-3 cells produce a ligand which binds tothe extracellular domain of Flt4 and activates this receptor.

EXAMPLE 5 Purification of the Flt4 Ligand

The ligand expressed by PC-3 cells as characterized in Example 4 waspurified and isolated using a recombinantly-produced Flt4 extracellulardomain (Flt4EC) in affinity chromatography.

Two harvests of serum-free conditioned medium, comprising a total of 8L, were collected from 500 confluent 15 cm diameter culture dishescontaining confluent layers of PC-3 cells. The conditioned medium wasclarified by centrifugation at 10,000×g and concentrated 80-fold usingan Ultrasette Tangential Flow Device (Filtron, Northborough, Mass.) witha 10 kD cutoff Omega Ultrafiltration membrane according to themanufacturer's instructions. Recombinant Flt4 extracellular domain wasexpressed in a recombinant baculovirus cell system and purified byaffinity chromatography on Ni-agarose (Ni-NTA affinity column obtainedfrom Qiagen). The purified extracellular domain was coupled toCNBr-activated Sepharose CL-4B at a concentration of 5 mg/ml and used asan affinity matrix for ligand affinity chromatography.

Concentrated conditioned medium was incubated with 2 ml of therecombinant Flt4 extracellular domain-Sepharose affinity matrix in arolling tube at room temperature for 3 hours. All subsequentpurification steps were at +4° C. The affinity matrix was thentransferred to a column (Pharmacia) with an inner diameter of 15 mm andwashed successively with 100 ml of PBS and 50 ml of 10 mM Na-phosphatebuffer (pH 6.8). Bound material was eluted step-wise with 100 mMglycine-HCl, successive 6 ml elutions having pHs of 4.0, 2.4, and 1.9.Several 2 ml fractions of the eluate were collected in tubes containing0.5 ml 1 M Na-phosphate (pH 8.0). Fractions were mixed immediately anddialyzed in 1 mM Tris-HCl (pH 7.5). Aliquots of 75 ul each were analyzedfor their ability to stimulate tyrosine phosphorylation of Flt4. Theultrafiltrate, 100 ul aliquots of the concentrated conditioned mediumbefore and after ligand affinity chromatography, as well as 15-foldconcentrated fractions of material released from the Flt4 extracellulardomain-Sepharose matrix during the washings were also analyzed for theirability to stimulate Flt4 tyrosine phosphorylation.

As shown in FIG. 6, lane 3, the concentrated conditioned medium inducedprominent tyrosine phosphorylation of Flt4 in transfected NIH 3T3 cellsoverexpressing Flt4. This activity was not observed in conditionedmedium taken after medium was exposed to the Flt4 Sepharose affinitymatrix described above (lane 4). The specifically-bound Flt4-stimulatingmaterial was retained on the affinity matrix upon washes in PBS, 10 mMNa-phosphate buffer (pH 6.8), and at pH 4.0 (lanes 5-7, respectively),and it was eluted in the first two 2 ml aliquots at pH 2.4 (lanes 8 and9). A further decrease of the pH of the elution buffer did not causerelease of additional Flt4-stimulating material (lane 11). Lane 1depicts a control wherein Flt4-expressing cells were treated withnonconditioned medium; lane 2 depicts results wherein Flt4-expressingcells were treated with the ultrafiltrate fraction of conditioned mediumcontaining polypeptides of less than 10 kD molecular weight.

Small aliquots of the chromatographic fractions were concentrated in aSpeedVac concentrator (Savant, Farmingdale, N.Y.) and subjected toSDS-PAGE under reducing conditions with subsequent silver staining ofthe gel. As shown in FIG. 7, the major polypeptide, having a molecularweight of approximately 23 kD (reducing conditions), was detected in thefractions containing Flt4 stimulating activity (corresponding to lanes 8and 9 in FIG. 6). That polypeptide was not found in the otherchromatographic fractions. On the other hand, all other componentsdetected in the two active fractions were also distributed in thestarting material and in small amounts in the other washing and elutionsteps after their concentration. Similar results were obtained in threeindependent affinity purifications, indicating that the 23 kDpolypeptide specifically binds to Flt4 and induces its tyrosinephosphorylation.

Fractions containing the 23 kD polypeptide were combined, dried in aSpeedVac concentrator and subjected to SDS-PAGE in a 12.5% gel. Theproteins from the gel were then electroblotted to Immobilon-P (PVDF)transfer membrane (Millipore, Marlborough, Mass.) and visualized bystaining of the blot with Coomassie blue R-250. The region containingonly the stained 23 kD band was cut from the blot and was subjected toN-terminal amino acid sequence analysis in a Prosite Protein SequencingSystem (Applied Biosystems, Foster City, Calif.). The data were analyzedusing a 610A Data Analysis System (Applied Biosystems). Analysisrevealed a single N-terminal sequence of NH₂-XEETIKFAAAHYNTEILK-COOH(SEQ ID NO: 13).

EXAMPLE 6 Construction of PC-3 cell cDNA Library in a EukaryoticExpression Vector.

Poly(A)⁺ RNA was isolated from five 15 cm diameter confluent dishes ofPC-3 cells by a single step method using oligo(dT) (Type III,Collaborative Research) cellulose affinity chromatography (Sambrook etal., Molecular Cloning, A Laboratory Manual; Cold Spring HarborLaboratory Press, 1989). The yield was 70 micrograms. Six micrograms ofthe Poly(A)⁺ RNA were used to prepare an oligo(dT)-primed cDNA libraryin the mammalian expression vector pcDNA I and the Librarian kit ofInvitrogen according to the instructions included in the kit. Thelibrary was estimated to contain about 10⁶ independent recombinants withan average insert size of approximately 1.8 kb.

EXAMPLE 7 Amplification of the Unique Nucleotide Sequence Encoding theFlt4 Ligand

Degenerate oligonucleotides were designed based on the N-terminal aminoacid sequence of the isolated Flt4 ligand and were used as primers in apolymerase chain reaction (PCR) to amplify cDNA encoding the Flt4 ligandfrom a PC-3 cell library. The overall strategy is schematically depictedin FIG. 9A, where the different primers have been marked with arrows.

The PCR was carried out using 1 microgram of DNA from the amplified PC-3cDNA library and a mixture of sense-strand primers comprising5′-GCAGARGARACNATHAA-3′ (SEQ ID NO: 14)(wherein R is A or G, N is A,G,Cor T and H is A, C or T), encoding amino acid residues 2-6 (EETIK, SEQID NO: 15) and antisense-strand primers 5′-GCAYTINARDATYTCNGT-3′ (SEQ IDNO: 16) (wherein Y is C or T and D is A, G or ), corresponding to aminoacid residues 14-18 (TEILK, SEQ ID NO: 17). Three extra nucleotides(GCA) were added to the 5′-terminus of each primer to increase annealingstability. Two successive PCR runs were carried out using 1 U perreaction of DynaZyme (F-500L, Finnzymes), a thermostable DNA polymerase,in a buffer supplied by the manufacturer (10 mM Tris-HCl, pH 8.8 at 25°C., 1.5 mM MgCl₂, 50 mM KCl, 0.1% Triton-X100), at an extensiontemperature of 72° C. The first PCR run was carried out for 43 cycles.The first three cycles were run at an annealing temperature of 33° C.for 2 minutes, and the remaining cycles were run at 42° C. for 1 minute.

The region of the gel containing a weak band of the expected size (57bp) was cut out from the gel and eluted. The eluted material wasreamplified for 30 cycles using the same primer pairs described above at42° C. for 1 minute. The amplified fragment was cloned into a pCR IIvector (Invitrogen) using the TA cloning kit (Invitrogen) and sequencedusing the radioactive dideoxynucleotide sequencing method of Sanger. Sixclones were analyzed and all contained the sequence encoding theexpected peptide (amino acid residues 2-18 of the Flt4 ligandprecursor). Nucleotide sequence spanning the region from the thirdnucleotide of codon 6 to the third nucleotide of codon 13 (the extensionregion) was identical in all six clones: 5′-ATTCGCTGCAGCACACTACAAC-3′(SEQ ID NO: 18) and thus was considered to represent an amplifiedproduct from the unique sequence encoding part of the amino terminus ofthe Flt4 ligand.

EXAMPLE 8

Amplification of the 5′-end of the cDNA Encoding the Flt4 Ligand

Based on the unique nucleotide sequence encoding the N-terminus of theisolated Flt4 ligand, two pairs of nested primers were designed toamplify, in two subsequent PCR-reactions, the complete 5′-end of thecorresponding cDNAs from one microgram of DNA from the above-describedPC-3 cDNA library. First, amplification was performed with primer5′-TCNGTGTTGTAGTGTGCTG-3′ (SEQ ID NO: 19), which is the antisense-strandprimer corresponding to amino acid residues 9-15 (AAHYNTE, SEQ ID NO:20), and sense-strand primer 5′-TAATACGACTCACTATAGGG-3′ (SEQ ID NO: 21),corresponding to the T7 RNA promoter of the pcDNAI vector used forconstruction of the library. “Touchdown” PCR was used as disclosed inDon, et al., Nucl. Acids Res., 19:4008 (1991), incorporated by referenceherein. The annealing temperature of the two first cycles was 62° C. andsubsequently the annealing temperature was decreased in every othercycle by 1° C. until a final temperature of 53° C. was reached, at whichtemperature 16 additional cycles were conducted. Annealing time was 1minute and extension at each cycle was conducted at 72° C. for 1 minute.Multiple amplified DNA fragments were obtained in the first reaction.The products of the first amplification (1 ul of a 1:100 dilution inwater) were used in the second amplification reaction employing thenested primers 5′-GTTGTAGTGTGCTGCAGCGAATTT-3′ (SEQ ID NO: 22), anantisense-strand primer corresponding to amino acid residues 6-13(KFAAAHYN, SEQ ID NO: 23) of the Flt4 ligand, and5′-TCACTATAGGGAGACCCAAGC-3′ (SEQ ID NO: 24), a sense-strand primercorresponding to nucleotides 2179-2199 of the pcDNAI vector. Thesequences of these sense and antisense primers overlapped with the 3′ends of the corresponding primers used in the first PCR. “Touchdown” PCRwas carried out by decreasing the annealing temperature from 72° C. to66° C. and continuing with 18 additional cycles at 66° C. The annealingtime was 1 minute and extension at each cycle was carried out at 72° C.for 2 minutes. One major product of about 220 bp and three minorproducts of about 270 bp, 150 bp, and 100 bp were obtained.

The amplified fragment of approximately 220 bp was cut out from theagarose gel, cloned into a pCRII vector using the TA cloning kit(Invitrogen) and sequenced. Three recombinant clones were analyzed andthey contained the sequence5′-TCACTATAGGGAGACCCAAGCTTGGTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGGTGGAATTCGACGAACTCATGACTGTACTCTACCCAGAATATTGGAAAATGTACAAGTGTCAGCTAAGGCAAGGAGGCTGGCAACATAACAGAGAACAGGCCAACCTCAACTCAAGGACAGAAGAGACTATAAAATTCGCTGCAGCACACTACAAC-3′ (SEQ ID NO: 25). The beginning ofthe sequence represents the pcDNAI vector and the underlined sequencerepresents the amplified product of the 5′-end of the insert.

EXAMPLE 9 Amplification of the 3′-end of cDNA Encoding the Flt4 Ligand

Based upon the amplified 5′-sequence of the clones encoding the aminoterminus of the 23 kD Flt4 ligand, two pairs of non-overlapping nestedprimers were designed to amplify the 3′-portion of theFlt-4-ligand-encoding cDNA clones. The sense-strand primer5′-ACAGAGAACAGGCCAACC-3′ (SEQ ID NO: 26) and antisense-strand primer5′-TCTAGCATTTAGGTGACAC-3′ (SEQ ID NO: 27) corresponding to nucleotides2311-2329 of the pcDNAI vector were used in a first “touchdown” PCR. Theannealing temperature of the reaction was decreased 1° C. every twocycles from 72° C. to 52° C., at which temperature 15 additional cycleswere carried out. The annealing time was 1 minute and extension at eachcycle was carried out at 72° C. for 3 minutes. DNA fragments of severalsizes were obtained in the first amplification. Those products werediluted 1:200 in water and reamplified in PCR using the second pair ofprimers: 5′-AAGAGACTATAAAATTCGCTGCAGC-3′ (SEQ ID NO: 28) and5′-CCCTCTAGATGCATGCTCGA-3′ (SEQ ID NO: 29) (antisense-strand primercorresponding to nucleotides 2279-2298 of the pcDNAI vector). Two DNAfragments were obtained, having sizes of 1350 bp and 570 bp. Thosefragments were cloned into a pCRII vector and the inserts of the cloneswere sequenced. Both of these fragments were found to contain sequencesencoding an amino acid sequence homologous to the VEGF sequence.

EXAMPLE 10 Screening the PC-3 Cell cDNA Library using the 5′ PCRFragment of Flt4 Ligand cDNA

A 219 bp 5′-terminal fragment of Flt4 ligand cDNA was amplified by PCRusing the 5′ PCR fragment described above and primers5′-GTTGTAGTGTGCTGCAGCGAATIT-3′ (antisense-strand primer, SEQ ID NO: 30)and 5′-TCACTATAGGGAGACCCAAGC-3′ (SEQ ID NO: 31) (sense-primercorresponding to nucleotides 2179-2199 of the pcDNAI vector). Theamplified product was subjected to digestion with EcoRI (BoehringerMannheim) to remove the portion of the DNA sequence amplified from thepcDNAI vector and the resulting 153 bp fragment encoding the 5′ end ofthe Flt4 ligand was labeled with [³²P]-dCTP using the Klenow fragment ofE. coli DNA polymerase I (Boehringer Mannheim). That fragment was usedas a probe for hybridization screening of the amplified PC-3 cell cDNAlibrary.

Filter replicas of the library were hybridized with the radioactivelylabeled probe at 42° C. for 20 hours in a solution containing 50%formamide, 5×SSPE, 5×Denhardt's solution, 0.1% SDS and 0.1 mg/mldenatured salmon sperm DNA. Filters were washed twice in 1×SSC, 0.1% SDSfor 30 minutes at room temperature, then twice for 30 minutes at 65° C.and exposed overnight.

On the basis of autoradiography, 10 positive recombinant bacterialcolonies hybridizing with the probe were chosen from the library.Plasmid DNA was purified from these colonies and analyzed by EcoRI andNotI digestion and agarose gel electrophoresis followed by ethidiumbromide staining. The ten plasmid clones were divided into three groupson the basis of the presence of insert sizes of approximately 1.7, 1.9and 2.1 kb, respectively. Inserts of plasmids from each group weresequenced using the T7 oligonucleotide as a primer and walking primersfor subsequent sequencing reactions.

Sequence analysis showed that all clones contain the open reading frameencoding the NH2-terminal sequence of the 23 kD Flt4 ligand. Dideoxysequencing was continued using walking primers in the downstreamdirection. A complete cDNA sequence and deduced amino acid sequence froma 2.1 kb clone is set forth in FIGS. 9B-9C (SEQ ID NOs: 32 and 33,respectively). A putative cleavage site of a “prepro” leader sequence isindicated in FIGS. 9B-9D with a shaded triangle. When compared withsequences in the GenBank Database, the predicted protein product of thisreading frame was found to be homologous with the predicted amino acidsequences of the PDGF/VEGF family of growth factors, as shown in FIGS.10A-10C.

Plasmid pFLT4-L, containing the 2.1 kb cDNA clone in pcDNAI vector, hasbeen deposited with the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md. 20852 as accession number 97231.

EXAMPLE 11 Stimulation of Flt4 Autophosphorylation by the ProteinProduct of the Flt4 Ligand Vector

The 2.1 kb cDNA insert of plasmid Flt4-L, which contains the openreading frame encoding the sequence shown in FIGS. 9B-9D (SEQ ID NO:32), was cut out from the pcDNAI vector using HindIII and NotIrestriction enzymes, isolated from a preparative agarose gel, andligated to the corresponding sites in the pREP7 expression vector(Invitrogen). The pREP7 vector containing the above cloned pFLT4-Linsert was transfected into 293-EBNA cells (Invitrogen) using thecalcium phosphate transfection method (Sambrook et al., MolecularCloning, A Laboratory Manual; Cold Spring Harbor Laboratory Press,1989). About 48 hours after transfection the medium of the transfectedcells was changed to DMEM medium lacking fetal calf serum and incubatedfor 36 h. The thus conditioned medium was then collected, centrifuged at5000×g for 20 minutes, the supernatant was concentrated 5-fold usingCentriprep 10 (Amicon) and used to stimulate NIH3T3 cells expressingLTRFlt4l, as in Example 4. The cells were lysed, immunoprecipitatedusing anti-Flt4 antiserum and analyzed by Western blotting usinganti-phosphotyrosine antibodies.

As can be seen from FIG. 11, lanes 1 and 3, the conditioned medium fromtwo different dishes of the transfected cells stimulated Flt4autophosphorylation in comparison with the medium from mock-transfectedcells, which gave only background levels of phosphorylation of the Flt4receptor (lane 2). When the concentrated conditioned medium waspre-absorbed with 20 microliters of a slurry of Flt4EC domain coupled toSepharose (see example 4), no phosphorylation was obtained (lane 4),showing that the activity responsible for Flt4 autophosphorylation wasindeed the Flt4 ligand. Thus, these results demonstrate that anexpression vector having an approximately 2.1 kb insert and containingan open reading frame as shown in FIGS. 9B-9D (and SEQ ID NO:32) isexpressed as a biologically active Flt4 ligand in transfected cells. Thesequence encoded by that open reading frame is shown in SEQ ID NO: 33.

The deduced molecular weight of a polypeptide consisting of the completeamino acid sequence in FIGS. 9B-9D (SEQ ID NO: 33, residues −102 to 317)is 46,883. The deduced molecular weight of a polypeptide consisting ofamino acid residues 1 to 317 of SEQ ID NO: 33 is 35,881. The Flt4 ligandpurified from PC-3 cultures had an observed molecular weight of about 23kD as assessed by SDS-PAGE under reducing conditions. Thus, it appearsthat the Flt4 ligand mRNA is translated into a precursor polypeptide,from which the mature ligand is derived by proteolytic cleavage. Also,the Flt4 ligand may be glycosylated at three putative N-linkedglycosylation sites conforming to the consensus which can be identifiedin the deduced Flt4 ligand amino acid sequence (N-residues underlined inFIGS. 10A-10C).

The carboxyl terminal amino acid sequences, which increase the predictedmolecular weight of the Flt4 ligand subunit in comparison with otherligands of this family, show a pattern of spacing of cysteine residuesreminiscent of the Balbiani ring 3 protein (BP3P) sequence (Dignam andCase, Gene, 88:133-140, 1990), as depicted schematically in FIG. 9A.Such a sequence may encode an independently folded domain present in aFlt4 ligand precursor and it may be involved, for example, in theregulation of secretion, solubility, stability, cell surfacelocalization or activity of the Flt4 ligand. Interestingly, at least onecysteine motif of the BR3P type is also found in the VEGF carboxyterminal amino acid sequences.

Thus, the Flt4 ligand mRNA appears first to be translated into aprecursor from the mRNA corresponding to the cDNA insert of plasmidFLT4-L, from which the mature ligand is derived by proteolytic cleavage.To define the mature Flt4 ligand polypeptide one first expresses thecDNA clone (which is deposited in the pcDNAI expression vector) incells, such as COS cells. One uses antibodies generated against encodedpeptides, fragments thereof, or bacterial Flt4 fusion proteins, such asa GST-fusion protein, to raise antibodies against the VEGF-homologousdomain and the amino- and carboxyl-terminal propeptides of Flt4 ligand.One then follows the biosynthesis and processing of the Flt4 ligand inthe transfected cells by pulse-chase analysis using radioactive cysteinefor labelling of the cells, immunoprecipitation and gel electrophoresis.Using antibodies against the three domains of the product encoded by thecDNA insert of plasmid FLT4-L, material for radioactive ornonradioactive amino-terminal sequence analysis is isolated. Thedetermination of the amino-terminal sequence of the mature VEGF-Cpolypeptide allows for identification of the amino-terminal proteolyticprocessing site. The determination of the amino-terminal sequence of thecarboxyl-terminal propeptide will give the carboxyl-terminal processingsite. This is confirmed by site-directed mutagenesis of the amino acidresidues adjacent to the cleavage sites, which would prevent thecleavage.

On the other hand, the Flt4 ligand is characterized by progressive 3′deletions in the 3′ coding sequences of the Flt4 ligand precursor clone,introducing a stop codon resulting in carboxy-terminal truncations ofits protein product. The activities of such truncated forms are assayzedby, for example, studying Flt4 autophosphorylation induced by thetruncated proteins when applied to cultures of cells, such as NIH 3T3cells expressing LTRFlt4. By extrapolation from studies of the structureof the related platelet derived growth factor (PDGF, reference Heldin etal., Growth Factors 8:245-252 (1993)) one determines that the regioncritical for receptor activation by the Flt4 ligand is contained withinits first approximately 180 amino acid residues of the secreted VEGF-Cprotein lacking the putative 102 amino acid prepro leader, andapparently within the first approximately 120 amino acid residues.

On the other hand, the difference between the molecular weights observedfor the purified ligand and deduced from the open reading frame of theFlt4 precursor clone may be due to the fact that the soluble ligand wasproduced from an alternatively spliced mRNA which would also be presentin the PC-3 cells, from which the isolated ligand was derived. Toisolate such alternative cDNA clones one uses cDNA fragments of thedeposited clone and PCR primers made according to the sequence providedas well as techniques standard in the art to isolate or amplifyalternative cDNAs from the PC-3 cell cDNA library. One may also amplifyusing reverse transcription (RT)-PCR directly from the PC-3 mRNA usingthe primers provided in the sequence of the cDNA insert of plasmidFLT4-L. Alternative cDNA sequences are determined from the resultingcDNA clones. One can also isolate genomic clones corresponding to theFlt4 ligand mRNA transcript from a human genomic DNA library usingmethods standard in the art and to sequence such clones or theirsubcloned fragments to reveal the corresponding exons. Alternative exonscan then be identified by a number of methods standard in the art, suchas heteroduplex analysis of cDNA and genomic DNA, which are subsequentlycharacterized.

EXAMPLE 12 Expression of the Gene Encoding VEGF-C in Human Tumor CellLines

Expression of transcripts corresponding to the Flt4 ligand (VEGF-C) wasanalyzed by hybridization of Northern blots containing isolated poly(A)⁺RNA from HT-1080 and PC-3 human tumor cell lines. The probe was theradioactively labelled insert of the 2.1 kb cDNA clone (specificactivity 10⁸-10⁹ cpm/mg of DNA). The blot was hybridized overnight at42° C. using 50% formamide, 5×SSPE buffer, 2% SDS, 10×Denhardt'ssolution, 100 mg/ml salmon sperm DNA and 1×10⁶ cpm of the labelledprobe/ml. The blot was washed at room temperature for 2×30 minutes in2×SSC containing 0.05% SDS, and then for 2×20 min at 52° C. in 0.1×SSCcontaining 0.1% SDS. The blot was then exposed at −70° C. for three daysusing intensifying screens and Kodak XAR film. Both cell lines expressedan Flt4 ligand mRNA of about 2.4 kb, as well as VEGF and VEGF-B mRNA's(FIG. 12).

EXAMPLE 13 VEGF-C Chains Are Proteolytically Processed afterBiosynthesis and Disulfide Linked

The predicted molecular mass of the secreted polypeptide, as deducedfrom the VEGF-C ORF, is 46,883 kD, suggesting that VEGF-C mRNA may befirst translated into a precursor, from which the mature ligand of 23 kDis derived by proteolytic cleavage.

To study this, metabolic labelling of 293 EBNA cells transfected withthe VEGF-C construct was carried out by addition of 100 mci/ml ofPro-mix™ L-[³⁵S] in vitro cell labelling mix (Amersham) to the culturemedium devoid of cysteine and methionine. After two hours, the celllayers were washed twice with PBS and the medium was then replaced withDMEM-0.2% BSA. After 1, 3, 6, 12 and 24 hours of subsequent incubation,the culture medium was collected, clarified by centrifugation, andconcentrated, and VEGF-C was bound to 30 microliters of a slurry ofFlt4EC-Sepharose overnight at +4° C., followed by three washes in PBS,two washes in 20 mM Tris-HCl (pH 7.5), alkylation, SDS-PAGE andautoradiography.

These experiments demonstrated that a putative precursor polypeptide of32 kD apparent molecular mass was bound to the Flt4EC affinity matrixfrom the CM of metabolically labelled cells transfected with a VEGF-Cexpression vector (FIG. 13A). Increased amounts of a 23 kD receptorbinding polypeptide accumulated in the culture medium during asubsequent chase period of 3 h, but not thereafter (lanes 2-4 and datanot shown), suggesting that the 23 kD form is produced by proteolyticprocessing, which is cell-associated and incomplete, at least in thetransiently transfected cells. The arrows in FIG. 13A indicate the 32kDa and 23 kDa polypeptides of secreted VEGF-C.

In a related experiment, VEGF-C isolated using Flt4EC-Sepharose after a4 h continuous metabolic labelling was analyzed by polyacrylamide gelelectrophoresis in nonreducing conditions (FIG. 13B). Higher molecularmass forms were observed under nonreducing conditions, suggesting thatthe VEGF-C polypeptides can form disulfide-linked dimers and/ormultimers (arrows in FIG. 13B).

EXAMPLE 14 Stimulation Of VEGFR-2 Autophosphorylation By VEGF-C

Conditioned medium (CM) from 293 EBNA cells transfected with the VEGF-Cvector also was used to stimulate porcine aortic endothelial (PAE) cellsexpressing VEGFR-2. Pajusola et al., Oncogene, 9:3545-55 (1994);Waltenberger et al., J. Biol. Chem., 269:26988-95 (1994). The cells werelysed and immunoprecipitated using VEGFR-2- specific antiserum(Waltenberger et al., 1994).

PAE-KDR cells (Waltenberger et al., 1994) were grown in Ham's F12medium-10% fetal calf serum (FCS). Confluent NIH 3T3 Flt4 cells orPAE-KDR cells were starved overnight in DMEM or Ham's F12 media,respectively, supplemented with 0.2% bovine serum albumin (BSA), andthen incubated for 5 min. with the analyzed media. Recombinant humanVEGF (R&D Systems) and PDGF-BB were used as a control stimulatingagents. The cells were washed twice with ice-cold tris-buffered saline(TBS) containing 100 mM sodium orthovanadate and lysed in RIPA buffercontaining 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.1 U/ml aprotininand 1 mM sodium orthovanadate. The lysates were sonicated, clarified bycentrifugation at 16,000 g for 20 min. and incubated for 3-6 h on icewith 3-5 microliters of antisera specific for Flt4 (Pajusola et al.,1993), VEGFR-2 or PDGFR-β (Claesson-Welsh et al., J. Biol. Chem.,264:1742-47 (1989); Waltenberger et al., 1994). Immunoprecipitates werebound to protein A-Sepharose, washed three times with RIPA buffercontaining 1 mM PMSF, 1 mM sodium orthovanadate, twice with 10 mMTris-HCl (pH 7.4) and subjected to SDS-PAGE in a 7% gel. Polypeptideswere transferred to nitrocellulose by Western blotting and analyzedusing PY20 phosphotyrosine-specific monoclonal antibodies (TransductionLaboratories) or receptor-specific antiserum and ECL method (Amersham).

The results of the experiment are presented in FIGS. 14A and 14B. Asshown in FIG. 14A, PAE cells expressing VEGFR-2 were stimulated with 10-or 2-fold concentrated medium from mock-transfected 293-EBNA cells(lanes 1 and 2), or with 2-, 5- or 10-fold concentrated medium from293-EBNA cell cultures expressing the recombinant VEGF-C (lanes 3-6).VEGFR-2 was immunoprecipitated with specific antibodies and analyzed bySDS-PAGE and Western blotting using phosphotyrosine antibodies. Forcomparison, the stimulations were carried out with non-conditionedmedium containing 50 ng/ml of purified recombinant VEGF (lanes 7 and 8).Lanes 6 and 7 show stimulation with VEGF-C- or VEGF-containing mediapretreated with Flt4EC. As depicted in FIG. 14B, PDGFR-β-expressing NIH3T3 cells were stimulated with non-conditioned medium (lane 1), 5-foldconcentrated CM from mock-transfected (lane 2) or VEGF-C-transfected(lanes 3 and 4) cells, or with non-conditioned medium containing 50ng/ml of recombinant human PDGF-BB (lane 5). Medium containing VEGF-Cwas also pretreated with recombinant Flt4EC (lane 4). PDGFR-β wasimmunoprecipitated with specific antibodies and analyzed by SDS-PAGE andWestern blotting using phosphotyrosine antibodies with subsequentstripping and reprobing of the membrane with antibodies specific forPDGFR-β.

Referring again to FIG. 14A, a basal level of tyrosine phosphorylationof VEGFR-2 was detected in cells stimulated by CM from themock-transfected cells. A further concentration of this medium resultedin only a slight enhancement of VEGFR-2 phosphorylation (lanes 1 and 2).CM containing recombinant VEGF-C stimulated tyrosine autophosphorylationof VEGFR-2 and the intensity of the autophosphorylated polypeptide bandwas increased upon concentration of the VEGF-C CM (lanes 3-5).Furthermore, the stimulating effect was abolished after pretreatment ofthe medium with the Flt4EC affinity matrix (compare lanes 1, 5 and 6).The maximal effect of VEGF-C in this assay was comparable to the effectof recombinant VEGF added to unconditioned medium at concentration of 50ng/ml (lane 8). Pretreatment of the medium containing VEGF with Flt4ECdid not abolish its stimulating effect on VEGFR-2 (compare lanes 7 and8). These results suggest that the VEGF-C expression vector encodes aligand not only for Flt4 (VEGFR-3), but also for VEGFR-2.

In order to further confirm that the stimulating effect of VEGF-C ontyrosine phosphorylation of VEGFR-3 and VEGFR-2 was receptor-specific,we analyzed the effect of VEGF-C on tyrosine phosphorylation of PDGFreceptor β (PDGFR-β) which is abundantly expressed on fibroblasticcells. As can be seen from FIG. 14B, a weak tyrosine phosphorylation ofPDGFR-β was detected upon stimulation of Flt4-expressing NIH 3T3 cellswith CM from the mock-transfected cells (compare lanes 1 and 2). Asimilar low level of PDGFR-β phosphorylation was observed when the cellswere incubated with CM from the VEGF-C transfected cells, with orwithout prior treatment with Flt4EC (lanes 3 and 4). In contrast, theaddition of 50 ng/ml of PDGF-BB induced a prominent tyrosineautophosphorylation of PDGFR-β (lane 5).

EXAMPLE 15 VEGF-C Stimulates Endothelial Cell Migration In Collagen Gel

CM from cell cultures transfected with the VEGF-C expression vector wasplaced in a well made in collagen gel and used to stimulate themigration of bovine capillary endothelial (BCE) cells in thethree-dimensional collagen gel as follows.

BCE cells (Folkman et al., Proc. Nat'l Acad. Sci. USA, 76:5217-5221(1979) were cultured as described in Pertovaara et al., J. Biol. Chem.,269:6271-74 (1994). The collagen gels were prepared by mixing type Icollagen stock solution (5 mg/ml in 1 mM HCl) with an equal volume of2×MEM and 2 volumes of MEM containing 10% newborn calf serum to give afinal collagen concentration of 1.25 mg/ml. The tissue culture plates (5cm diameter) were coated with about 1 mm thick layer of the solution,which was allowed to polymerize at 37° C. BCE cells were seeded on topof this layer. For the migration assays, the cells were allowed toattach inside a plastic ring (1 cm diameter) placed on top of the firstcollagen layer. After 30 min., the ring was removed and unattached cellswere rinsed away. A second layer of collagen and a layer of growthmedium (5% newborn calf serum (NCS)), solidified by 0.75% low meltingpoint agar (FMC BioProducts, Rockland, ME), were added. A well (3 mmdiameter) was punched through all the layers on both sides of the cellspot at a distance of 4 mm, and the sample or control media werepipetted daily into the wells. Photomicrographs of the cells migratingout from the spot edge were taken after six days through an Olympus CK 2inverted microscope equipped with phase-contrast optics. The migratingcells were counted after nuclear staining with the fluorescent dyebisbenzimide (1 mg/ml, Hoechst 33258, Sigma).

FIG. 15A depicts a comparison of the number of cells migrating atdifferent distances from the original area of attachment towards wellscontaining media conditioned by the non-transfected (control) ortransfected (mock; VEGF-C; VEGF) cells, 6 days after addition of themedia. The number of cells migrating out from the original ring ofattachment was counted in five adjacent 0.5 mm×0.5 mm squares using amicroscope ocular lens grid and 10× magnification. Cells migratingfurther than 0.5 mm were counted in a similar way by moving the grid in0.5 mm steps. The experiments were carried out twice with similarresults, and medium values from the one of the experiments are presentedwith standard error bars. The photographs in FIGS. 15B-15E depictphase-contrast microscopy and fluorescent microscopy of the nuclearstaining of BCE cells migrating towards the wells containing mediaconditioned by the mock-transfected cells or by VEGF-C-transfectedcells. The areas shown is approximately 1 mm×1.5 mm, and arrows indicatethe borders of the original ring of attachment.

After 6 days of treatment, the cultures were stained and cells atdifferent distances outside of the original ring of attachment werecounted using fluorescent nuclear staining and detection with afluorescence microscope equipped with a grid. A comparison of thenumbers of migrating cells in successive 0.5 mm×0.5 mm areas is shown inFIG. 15A. As can be seen from the columns, VEGF-C-containing CMstimulated cell migration more than medium conditioned by thenon-transfected or mock-transfected cells but less than medium fromcells transfected with a VEGF expression vector. An example of typicalphase contrast and fluorescent microscopic fields of cultures stimulatedwith medium from mock-transfected or VEGF-C transfected cells is shownin FIGS. 15B-15E. Daily addition of 1 ng of FGF2 into the wells resultedin the migration of approximately twice the number of cells whencompared to the stimulation by CM from VEGF-transfected cells.

EXAMPLE 16 VEGF-C Is Expressed In Multiple Tissues

Northern blots containing 2 micrograms of isolated poly(A)⁺ RNA frommultiple human tissues (blot from Clontech) were probed withradioactively labelled insert of the 2.1 kb VEGF-C cDNA clone. Northernblotting and hybridization analysis showed that the 2.4 kb RNA andsmaller amounts of a 2.0 kb mRNA are expressed in multiple humantissues, most prominently in the heart, placenta, muscle, ovary andsmall intestine (FIG. 16A). Very little VEGF-C RNA was seen in thebrain, liver or thymus and peripheral blood leukocytes (pbl) appearednegative. A similar analysis of RNA from human fetal tissues (FIG. 16B)shows that VEGF-C is highly expressed in the kidney and lung and to alesser degree in the liver, while essentially no expression is detectedin the brain. Interestingly, VEGF expression correlates with VEGF-Cexpression in these tissues, whereas VEGF-B is highly expressed in alltissues analyzed.

EXAMPLE 17 The VEGF-C Gene Localizes To Chromosome 4q34

A DNA panel of 24 interspecies somatic cell hybrids, which had retainedone or two human chromosomes, was used for the chromosomal localizationof the VEGF-C gene (Bios Laboratories, Inc., New Haven, Conn.). Primerswere designed to amplify an about 250 bp fragment of the VEGF-C genefrom somatic cell hybrid DNA. The primers and conditions for polymerasechain reaction (PCR) were 5′-TGAGTGATTTGTAGCTGCTGTG-3′ (forward) [SEQ IDNO:34] and 5′-TATTGCAGCAACCCCCACATCT-3′ (reverse) [SEQ ID NO:35] forVEGF-C (94° C., 60s/62° C., 45s/72° C., 60s). The PCR products wereevaluated by electrophoresis in 1% agarose gels and visualized byethidium bromide staining in ultraviolet light. [α-³²P]-dCTP-labelledcDNA inserts of a plasmid representing the complete VEGF-C coding domainwas used as a probe in Southern blotting and hybridization analysis ofthe somatic cell hybrid DNAs as instructed by the supplier (BiosLaboratories).

The cell lines for fluorescence in situ hybridization (FISH) wereobtained from the American Type Culture Collection (Rockville, Md.).Purified DNA from P1 clones 7660 and 7661 (VEGF-C) (Genome Systems,Inc., St. Louis, Mo.) were confirmed positive by Southern blotting ofEco RI-digested DNA followed by hybridization with the VEGF-C cDNA. TheP1 clones were then labelled by nick translation either withbiotin-11-dUTP, biotin-14-ATP (Sigma Chemical Co., St. Louis, Mo.) ordigoxigenin 11-dUTP (Boehringer Mannheim GmbH, Mannheim, Germany)according to standard protocols. PHA-stimulated peripheral bloodlymphocyte cultures were treated with 5-bromodeoxyuridine (BrdU) at anearly replicating phase to induce G-banding. See Takahashi et al., HumanGenet., 86:14-16 (1995); Lemieux et al., Cytogenet. Cell Genet.,59:311-12 (1992). The FISH procedure was carried out in 50% formamide,10% dextran sulphate in 2×SSC using well-known procedures. See, e.g.,Rytkönnen et al., Cytogenet, Cell Genet., 68:61-63 (1995); Lichter etal., Proc. Natl. Acad. Sci. USA, 85:9664-68 (1988). Repetitive sequenceswere suppressed with 50-fold excess of Cot-1 DNA (BRL, Gaithenburg, Md.)compared with the labeled probe. Specific hybridization signals weredetected by incubating the hybridized slides in labelled antidigoxigeninantibodies, followed by counterstaining with 0.1 mmol/L4,6-diamino-2-phenylindole. Probe detection for two-color experimentswas accomplished by incubating the slides in fluorescein isothiocyanate(FITC)-anti-digoxigenin antibodies (Sigma Chemical Co.) and Texasred-avidin (Vector Laboratories, Burlingame, Calif.) orrhodamine-anti-digoxigenin and FITC-avidin.

Multi-color digital image analysis was used for acquisition, display andquantification of hybridization signals of metaphase chromosomes. Thesystem contains a PXL camera (Photometrics Inc., Tucson, Ariz.) attachedto a PowerMac 7100/Av workstation. IPLab software controls the cameraoperation, image acquisition and Ludl Filter wheel. At least 50 nucleiwere scored. Overlapping nuclei and clusters of cells were ignored. Aslide containing normal lymphocyte metaphase spreads and interphasenuclei was included in each experiment to control for the efficiency andspecificity of the hybridization.

In order to determine the chromosomal localization of the human VEGF-Cgene, DNAs from human rodent somatic cell hybrids containing definedsets of human chromosomes were analyzed by Southern blotting andhybridization with the VEGF-C cDNA probe. Among 24 DNA samples on thehybrid panel, representing different human chromosomes, human-specificsignals were observed only in hybrids which contained human chromosome4. The results were confirmed by PCR of somatic cell hybrid DNA usingVEGF-C specific primers, where amplified bands were obtained only fromDNAs containing human chromosome 4.

A genomic P1 plasmid for VEGF-C was isolated using specific primers andPCR and verified by Southern blotting and hybridization using a VEGF-Cspecific cDNA probe. The chromosomal localization of VEGF-C was furtherstudied using metaphase FISH. Using the P1 probe for VEGF-C in FISH aspecific hybridization to the 4q34 chromosomal band was detected in 40out of 44 metaphases (FIG. 17). Double-fluorochrome hybridization usinga cosmid probe specific for the aspartylglucosaminidase (AGA) geneshowed that VEGF-C is located just proximal to the AGA gene previouslymapped to the 4q34-35 chromosomal band.

Biotin labelled VEGF-C P1 and digoxigenin. labeled AGA cosmid probeswere hybridized simultaneously to metaphase chromosomes. This experimentdemonstrated that the AGA gene is more telomerically located than theVEGF-C gene. The foregoing example demonstrates the utility ofpolynucleotides of the invention as chromosomal markers.

EXAMPLE 18 Effect of Glucose Concentration and Hypoxia on VEGF, VEGF-Band VEGF-C mRNA Levels in C6 Glioblastoma Cells

Confluent cultures of C6 cells (ATCC CCL 107) were grown on 10 cmdiameter tissue culture plates containing 2.5 ml of DMEM and 5% fetalcalf serum plus antibiotics. The cultures were exposed for 16 hours tonormoxia in a normal cell culture incubator containing 5% CO₂ (FIG. 18:lanes marked −) or hypoxia (FIG. 18: lanes marked +) by closing theculture plates in an airtight glass chamber and burning a piece of woodinside until the flame was extinguished due to lack of oxygen.Polyadenylated RNA was isolated (as in the other examples), and 8micrograms of the RNA was electrophoresed and blot-hybridized with amixture of the VEGF, VEGF-B and VEGF-C probes (see FIG. 12). The resultsshow that hypoxia strongly induces VEGF (VEGF-A) mRNA expression(compare lanes − and +), both in low and high glucose, but has nosignificant effect on the VEGF-B mRNA levels. The VEGF-C mRNA isolatedfrom hypoxic cells runs slightly faster in gel electrophoresis and anextra band of faster mobility can be seen below the upper mRNA band.This observation suggests that hypoxia affects VEGF-C RNA processing.One explanation for this observation is that VEGF-C mRNA splicing isaltered, affecting the VEGF-C open reading frame and resulting in analternative VEGF-C protein being produced by hypoxic cells. Suchalternative forms of VEGF-C and VEGF-C-encoding polynucleotides arecontemplated as an aspect of the invention. This data indicatesscreening and diagnostic utilities for polynucleotides and polypeptidesof the invention, such as methods whereby a biological sample isscreened for the hypoxia-induced form of VEGF-C and/or VEGF-C mRNA. Thedata further suggests a therapeutic indication for antibodies and/orother inhibitors of the hypoxia-induced form of VEGF-C or the normalform of VEGF-C.

EXAMPLE 19 Pulse-chase Labeling and Immunoprecipitation of VEGF-CPolypeptides from 293 Cells Transfected with VEGF-C Expression Vector.

The following VEGF-C branched amino-terminal peptide, designated PAM126,was synthesized for production of anti-VEGF-C antiserum:

NH₂-E-E-T-I-K-F-A-A-A-H-Y-N-T-E-I-L-K-COOH (SEQ ID NO: 39). Inparticular, PAM126 was synthesized as a branched polylysine structureK3PA4 having four peptide acid (PA) chains attached to two availablelysine (K) residues. The synthesis was performed on a 433A PeptideSynthesizer (Applied Biosystems) using Fmoc-chemistry and TentaGel S MAPRAM10 resin mix (RAPP Polymere GmbH, Tubingen, Germany), yielding bothcleavable and resin-bound peptides. The cleavable peptide was purifiedvia reverse phase HPLC and was used together with the resin-boundpeptide in immunizations. The correctness of the synthesis products wereconfirmed using mass-spectroscopy (Lasermatt).

The peptide was dissolved in phosphate buffered saline (PBS), mixed withFreund's adjuvant, and used for immunization of rabbits at bi-weeklyintervals using methods standard in the art (Harlow and Lane,Antibodies, a laboratory manual, Cold Spring Harbor Laboratory Press(1988)). Antisera obtained after the fourth booster immunization wasused for immunoprecipitation of VEGF-C in pulse-chase experiments, asdescribed below.

For pulse-chase analysis, 293 cells transfected with a VEGF-C expressionvector (i.e., the FLT4-L cDNA inserted into the pREP7 expression vectoras described above) were incubated for 30 minutes in methionine-free,cysteine-free, serum-free DMEM culture medium at 37° C. The medium wasthen changed, and 200 microCuries of ³⁵S-methionine and cysteine(Promix, Amersham, Buckinghamshire, England) was added. The cell layerswere incubated in this labeling medium for two hours, washed with PBS,and incubated for 0, 15, 30, 60, 90, 120, or 180 minutes in serum-freeDMEM (chase). After the various chase periods, the medium was collected,the cells were again washed two times in PBS, and lysed inimmunoprecipitation buffer. The VEGF-C polypeptides were analyzed fromboth the culture medium and from the cell lysates byimmunoprecipitation, using the VEGF-C-specific antiserum raised againstthe NH₂-terminal peptide (PAM126) of the 23 kD VEGF-C form.Immunoprecipitated polypeptides were analyzed via SDS-PAGE followed byautoradiography.

Referring to FIGS. 19A and 19B, the resultant autoradiograms demonstratethat immediately after a 2 hour labeling (chase time 0), the VEGF-Cvector-transfected cells contained a radioactive 55 kD polypeptide band,which is not seen in mock-transfected cells (M). This 55 kD polypeptideband gradually diminishes in intensity with increasing chase periods,and is no longer detected in the cells by 180 minutes of chase. A 32 kDpolypeptide band also is observed in VEGF-C transfected cells (and notmock-transfected cells). This 32 kD band disappears with similarkinetics to that of the 55 kD band. Simultaneously, increasing amountsof 32 kD (arrow) and subsequently 23 kD (arrow) and 14 kD polypeptidesappear in the medium.

Collectively, the data from the pulse-chase experiments indicate thatthe 55 kD intracellular polypeptide represents a pro-VEGF-C polypeptide,which is not secreted from cells, but rather is first proteolyticallycleaved into the 32 kD form. The 32 kD form is secreted andsimultaneously further processed by proteolysis into the 23 kD and 14 kdforms. Without intending to be limited to a particular theory, it isbelieved that processing of the VEGF-C precursor occurs as removal of asignal sequence, removal of the COOH-terminal domain (BR3P), and removalof an amino terminal peptide, resulting in a VEGF-C polypeptide havingthe TEE . . . amino terminus.

At high resolution, the 23 kD polypeptide band appears as a closelyspaced polypeptide doublet, suggesting heterogeneity in cleavage orglycosylation.

EXAMPLE 20 Isolation of Mouse cDNA Clones Encoding VEGF-C

To clone a mouse variant of VEGF-C, approximately 1×10⁶ bacteriophagelambda clones of a commercially-available 12 day mouse embryonal cDNAlibrary (lambda EXlox library, Novagen # 69632-1) were screened with aradiolabeled fragment of human VEGF-C cDNA containing nucleotides 495 to1661 of SEQ ID NO:32. One positive clone was isolated.

A 1323 bp EcoRI/HindIII fragment of the insert of the isolated mousecDNA clone was subcloned into the corresponding sites of the pBluescriptSK+vector (Stratagene) and sequenced. The cDNA sequence of this clonewas homologous to the human VEGF-C sequence reported herein, except thatabout 710 bp of 5′-end sequence present in the human clone was notpresent in the mouse clone.

For further screening of mouse cDNA libraries, a HindIII-BstXI (Hind IIIsite is from the pBluescript SK+ polylinker) fragment of 881 bp from thecoding region of the mouse cDNA clone was radiolabeled and used as aprobe to screen two additional mouse cDNA libraries. Two additional cDNAclones from an adult mouse heart ZAP II cDNA library (Stratagene #936306) were identified. Three additional clones also were isolated froma mouse heart 5′-stretch-plus cDNA library in λgt11 (Clontech #ML5002b).Of the latter three clones, one was found to contain an insert of about1.9 kb. The insert of this cDNA clone was subcloned into EcoRI sites ofpBluescript SK+ vector and both strands of this clone were completelysequenced, resulting in the nucleotide and deduced amino acid sequencesshown in SEQ ID NOs: 40 and 41.

It is contemplated that the polypeptide corresponding to SEQ ID NO: 41is processed into a mature mouse VEGF-C protein, in a manner analogousto the processing of the human VEGF-C prepropeptide. Putative cleavagesites for the mouse protein are identified using procedures outlinedabove for identification of cleavage sites for the human VEGF-Cpolypeptide.

The foregoing example demonstrates the utility of polynucleotides of theinvention for identifying and isolating polynucleotides encoding othernon-human mammalian variants of VEGF-C. Such identified and isolatedpolynucleotides, in turn, can be expressed (using procedures similar tothose described in preceding examples) to produce recombinantpolypeptides corresponding to non-human mammalian variants of VEGF-C.

Deposit of Biological Materials

Plasmid FLT4-L has been deposited with the American Type CultureCollection (ATCC), 12301 Parklawn Dr., Rockville Md. 20952 (USA),pursuant to the provisions of the Budapest Treaty, and has been assigneda deposit date of Jul. 24, 1995 and ATCC accession number 97231.

While the present invention has been described in terms of specificembodiments, it is understood that variations and modifications willoccur to those in the art. Accordingly, only such limitations as appearin the appended claims should be placed on the invention.

41 20 base pairs nucleic acid single linear DNA (genomic) 1 TGTCCTCGCTGTCCTTGTCT 20 70 base pairs nucleic acid single linear DNA (genomic) 2ACATGCATGC CACCATGCAG CGGGGCGCCG CGCTGTGCCT GCGACTGTGG CTCTGCCTGG 60GACTCCTGGA 70 24 base pairs nucleic acid single linear DNA (genomic) 3ACATGCATGC CCCGCCGGTC ATCC 24 22 base pairs nucleic acid single linearDNA (genomic) 4 CGGAATTCCC CATGACCCCA AC 22 33 base pairs nucleic acidsingle linear DNA (genomic) 5 CCATCGATGG ATCCTACCTG AAGCCGCTTT CTT 33 17base pairs nucleic acid single linear DNA (genomic) 6 ATTTAGGTGA CACTATA17 34 base pairs nucleic acid single linear DNA (genomic) 7 CCATCGATGGATCCCGATGC TGCTTAGTAG CTGT 34 40 amino acids amino acid single linearprotein 8 Pro Met Thr Pro Thr Thr Tyr Lys Gly Ser Val Asp Asn Gln ThrAsp 1 5 10 15 Ser Gly Met Val Leu Ala Ser Glu Glu Phe Glu Gln Ile GluSer Arg 20 25 30 His Arg Gln Glu Ser Gly Phe Arg 35 40 21 base pairsnucleic acid single linear DNA (genomic) 9 CTGGAGTCGA CTTGGCGGAC T 21 60base pairs nucleic acid single linear DNA (genomic) 10 CGCGGATCCCTAGTGATGGT GATGGTGATG TCTACCTTCG ATCATGCTGC CCTTATCCTC 60 34 base pairsnucleic acid single linear DNA (genomic) 11 CCCAAGCTTG GATCCAAGTGGCTACTCCAT GACC 34 20 base pairs nucleic acid single linear DNA(genomic) 12 GTTGCCTGTG ATGTGCACCA 20 18 amino acids amino acid singlelinear peptide 13 Xaa Glu Glu Thr Ile Lys Phe Ala Ala Ala His Tyr AsnThr Glu Ile 1 5 10 15 Leu Lys 17 base pairs nucleic acid single linearDNA (genomic) 14 GCAGARGARA CNATHAA 17 5 amino acids amino acid singlelinear peptide 15 Glu Glu Thr Ile Lys 1 5 18 base pairs nucleic acidsingle linear DNA (genomic) 16 GCAYTTNARD ATYTCNGT 18 5 amino acidsamino acid single linear peptide 17 Thr Glu Ile Leu Lys 1 5 22 basepairs nucleic acid single linear DNA (genomic) 18 ATTCGCTGCA GCACACTACAAC 22 19 base pairs nucleic acid single linear DNA (genomic) 19TCNGTGTTGT AGTGTGCTG 19 7 amino acids amino acid single linear peptide20 Ala Ala His Tyr Asn Thr Glu 1 5 20 base pairs nucleic acid singlelinear DNA (genomic) 21 TAATACGACT CACTATAGGG 20 24 base pairs nucleicacid single linear DNA (genomic) 22 GTTGTAGTGT GCTGCAGCGA ATTT 24 8amino acids amino acid single linear peptide 23 Lys Phe Ala Ala Ala HisTyr Asn 1 5 21 base pairs nucleic acid single linear DNA (genomic) 24TCACTATAGG GAGACCCAAG C 21 219 base pairs nucleic acid single linear DNA(genomic) 25 TCACTATAGG GAGACCCAAG CTTGGTACCG AGCTCGGATC CACTAGTAACGGCCGCCAGT 60 GTGGTGGAAT TCGACGAACT CATGACTGTA CTCTACCCAG AATATTGGAAAATGTACAAG 120 TGTCAGCTAA GGCAAGGAGG CTGGCAACAT AACAGAGAAC AGGCCAACCTCAACTCAAGG 180 ACAGAAGAGA CTATAAAATT CGCTGCAGCA CACTACAAC 219 18 basepairs nucleic acid single linear DNA (genomic) 26 ACAGAGAACA GGCCAACC 1819 base pairs nucleic acid single linear DNA (genomic) 27 TCTAGCATTTAGGTGACAC 19 25 base pairs nucleic acid single linear DNA (genomic) 28AAGAGACTAT AAAATTCGCT GCAGC 25 20 base pairs nucleic acid single linearDNA (genomic) 29 CCCTCTAGAT GCATGCTCGA 20 24 base pairs nucleic acidsingle linear DNA (genomic) 30 GTTGTAGTGT GCTGCAGCGA ATTT 24 21 basepairs nucleic acid single linear DNA (genomic) 31 TCACTATAGG GAGACCCAAGC 21 1997 base pairs nucleic acid single linear DNA (genomic) CDS352..1608 mat_peptide 658..1608 32 CCCGCCCCGC CTCTCCAAAA AGCTACACCGACGCGGACCG CGGCGGCGTC CTCCCTCGCC 60 CTCGCTTCAC CTCGCGGGCT CCGAATGCGGGGAGCTCGGA TGTCCGGTTT CCTGTGAGGC 120 TTTTACCTGA CACCCGCCGC CTTTCCCCGGCACTGGCTGG GAGGGCGCCC TGCAAAGTTG 180 GGAACGCGGA GCCCCGGACC CGCTCCCGCCGCCTCCGGCT CGCCCAGGGG GGGTCGCCGG 240 GAGGAGCCCG GGGGAGAGGG ACCAGGAGGGGCCCGCGGCC TCGCAGGGGC GCCCGCGCCC 300 CCACCCCTGC CCCCGCCAGC GGACCGGTCCCCCACCCCCG GTCCTTCCAC C ATG CAC 357 Met His -102 TTG CTG GGC TTC TTC TCTGTG GCG TGT TCT CTG CTC GCC GCT GCG CTG 405 Leu Leu Gly Phe Phe Ser ValAla Cys Ser Leu Leu Ala Ala Ala Leu -100 -95 -90 -85 CTC CCG GGT CCT CGCGAG GCG CCC GCC GCC GCC GCC GCC TTC GAG TCC 453 Leu Pro Gly Pro Arg GluAla Pro Ala Ala Ala Ala Ala Phe Glu Ser -80 -75 -70 GGA CTC GAC CTC TCGGAC GCG GAG CCC GAC GCG GGC GAG GCC ACG GCT 501 Gly Leu Asp Leu Ser AspAla Glu Pro Asp Ala Gly Glu Ala Thr Ala -65 -60 -55 TAT GCA AGC AAA GATCTG GAG GAG CAG TTA CGG TCT GTG TCC AGT GTA 549 Tyr Ala Ser Lys Asp LeuGlu Glu Gln Leu Arg Ser Val Ser Ser Val -50 -45 -40 GAT GAA CTC ATG ACTGTA CTC TAC CCA GAA TAT TGG AAA ATG TAC AAG 597 Asp Glu Leu Met Thr ValLeu Tyr Pro Glu Tyr Trp Lys Met Tyr Lys -35 -30 -25 TGT CAG CTA AGG AAAGGA GGC TGG CAA CAT AAC AGA GAA CAG GCC AAC 645 Cys Gln Leu Arg Lys GlyGly Trp Gln His Asn Arg Glu Gln Ala Asn -20 -15 -10 -5 CTC AAC TCA AGGACA GAA GAG ACT ATA AAA TTT GCT GCA GCA CAT TAT 693 Leu Asn Ser Arg ThrGlu Glu Thr Ile Lys Phe Ala Ala Ala His Tyr 1 5 10 AAT ACA GAG ATC TTGAAA AGT ATT GAT AAT GAG TGG AGA AAG ACT CAA 741 Asn Thr Glu Ile Leu LysSer Ile Asp Asn Glu Trp Arg Lys Thr Gln 15 20 25 TGC ATG CCA CGG GAG GTGTGT ATA GAT GTG GGG AAG GAG TTT GGA GTC 789 Cys Met Pro Arg Glu Val CysIle Asp Val Gly Lys Glu Phe Gly Val 30 35 40 GCG ACA AAC ACC TTC TTT AAACCT CCA TGT GTG TCC GTC TAC AGA TGT 837 Ala Thr Asn Thr Phe Phe Lys ProPro Cys Val Ser Val Tyr Arg Cys 45 50 55 60 GGG GGT TGC TGC AAT AGT GAGGGG CTG CAG TGC ATG AAC ACC AGC ACG 885 Gly Gly Cys Cys Asn Ser Glu GlyLeu Gln Cys Met Asn Thr Ser Thr 65 70 75 AGC TAC CTC AGC AAG ACG TTA TTTGAA ATT ACA GTG CCT CTC TCT CAA 933 Ser Tyr Leu Ser Lys Thr Leu Phe GluIle Thr Val Pro Leu Ser Gln 80 85 90 GGC CCC AAA CCA GTA ACA ATC AGT TTTGCC AAT CAC ACT TCC TGC CGA 981 Gly Pro Lys Pro Val Thr Ile Ser Phe AlaAsn His Thr Ser Cys Arg 95 100 105 TGC ATG TCT AAA CTG GAT GTT TAC AGACAA GTT CAT TCC ATT ATT AGA 1029 Cys Met Ser Lys Leu Asp Val Tyr Arg GlnVal His Ser Ile Ile Arg 110 115 120 CGT TCC CTG CCA GCA ACA CTA CCA CAGTGT CAG GCA GCG AAC AAG ACC 1077 Arg Ser Leu Pro Ala Thr Leu Pro Gln CysGln Ala Ala Asn Lys Thr 125 130 135 140 TGC CCC ACC AAT TAC ATG TGG AATAAT CAC ATC TGC AGA TGC CTG GCT 1125 Cys Pro Thr Asn Tyr Met Trp Asn AsnHis Ile Cys Arg Cys Leu Ala 145 150 155 CAG GAA GAT TTT ATG TTT TCC TCGGAT GCT GGA GAT GAC TCA ACA GAT 1173 Gln Glu Asp Phe Met Phe Ser Ser AspAla Gly Asp Asp Ser Thr Asp 160 165 170 GGA TTC CAT GAC ATC TGT GGA CCAAAC AAG GAG CTG GAT GAA GAG ACC 1221 Gly Phe His Asp Ile Cys Gly Pro AsnLys Glu Leu Asp Glu Glu Thr 175 180 185 TGT CAG TGT GTC TGC AGA GCG GGGCTT CGG CCT GCC AGC TGT GGA CCC 1269 Cys Gln Cys Val Cys Arg Ala Gly LeuArg Pro Ala Ser Cys Gly Pro 190 195 200 CAC AAA GAA CTA GAC AGA AAC TCATGC CAG TGT GTC TGT AAA AAC AAA 1317 His Lys Glu Leu Asp Arg Asn Ser CysGln Cys Val Cys Lys Asn Lys 205 210 215 220 CTC TTC CCC AGC CAA TGT GGGGCC AAC CGA GAA TTT GAT GAA AAC ACA 1365 Leu Phe Pro Ser Gln Cys Gly AlaAsn Arg Glu Phe Asp Glu Asn Thr 225 230 235 TGC CAG TGT GTA TGT AAA AGAACC TGC CCC AGA AAT CAA CCC CTA AAT 1413 Cys Gln Cys Val Cys Lys Arg ThrCys Pro Arg Asn Gln Pro Leu Asn 240 245 250 CCT GGA AAA TGT GCC TGT GAATGT ACA GAA AGT CCA CAG AAA TGC TTG 1461 Pro Gly Lys Cys Ala Cys Glu CysThr Glu Ser Pro Gln Lys Cys Leu 255 260 265 TTA AAA GGA AAG AAG TTC CACCAC CAA ACA TGC AGC TGT TAC AGA CGG 1509 Leu Lys Gly Lys Lys Phe His HisGln Thr Cys Ser Cys Tyr Arg Arg 270 275 280 CCA TGT ACG AAC CGC CAG AAGGCT TGT GAG CCA GGA TTT TCA TAT AGT 1557 Pro Cys Thr Asn Arg Gln Lys AlaCys Glu Pro Gly Phe Ser Tyr Ser 285 290 295 300 GAA GAA GTG TGT CGT TGTGTC CCT TCA TAT TGG AAA AGA CCA CAA ATG 1605 Glu Glu Val Cys Arg Cys ValPro Ser Tyr Trp Lys Arg Pro Gln Met 305 310 315 AGC TAAGATTGTACTGTTTTCCA GTTCATCGAT TTTCTATTAT GGAAAACTGT 1658 Ser GTTGCCACAGTAGAACTGTC TGTGAACAGA GAGACCCTTG TGGGTCCATG CTAACAAAGA 1718 CAAAAGTCTGTCTTTCCTGA ACCATGTGGA TAACTTTACA GAAATGGACT GGAGCTCATC 1778 TGCAAAAGGCCTCTTGTAAA GACTGGTTTT CTGCCAATGA CCAAACAGCC AAGATTTTCC 1838 TCTTGTGATTTCTTTAAAAG AATGACTATA TAATTTATTT CCACTAAAAA TATTGTTTCT 1898 GCATTCATTTTTATAGCAAC AACAATTGGT AAAACTCACT GTGATCAATA TTTTTATATC 1958 ATGCAAAATATGTTTAAAAT AAAATGAAAA TTGTATTAT 1997 419 amino acids amino acid linearprotein 33 Met His Leu Leu Gly Phe Phe Ser Val Ala Cys Ser Leu Leu AlaAla -102 -100 -95 -90 Ala Leu Leu Pro Gly Pro Arg Glu Ala Pro Ala AlaAla Ala Ala Phe -85 -80 -75 Glu Ser Gly Leu Asp Leu Ser Asp Ala Glu ProAsp Ala Gly Glu Ala -70 -65 -60 -55 Thr Ala Tyr Ala Ser Lys Asp Leu GluGlu Gln Leu Arg Ser Val Ser -50 -45 -40 Ser Val Asp Glu Leu Met Thr ValLeu Tyr Pro Glu Tyr Trp Lys Met -35 -30 -25 Tyr Lys Cys Gln Leu Arg LysGly Gly Trp Gln His Asn Arg Glu Gln -20 -15 -10 Ala Asn Leu Asn Ser ArgThr Glu Glu Thr Ile Lys Phe Ala Ala Ala -5 1 5 10 His Tyr Asn Thr GluIle Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys 15 20 25 Thr Gln Cys Met ProArg Glu Val Cys Ile Asp Val Gly Lys Glu Phe 30 35 40 Gly Val Ala Thr AsnThr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr 45 50 55 Arg Cys Gly Gly CysCys Asn Ser Glu Gly Leu Gln Cys Met Asn Thr 60 65 70 Ser Thr Ser Tyr LeuSer Lys Thr Leu Phe Glu Ile Thr Val Pro Leu 75 80 85 90 Ser Gln Gly ProLys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser 95 100 105 Cys Arg CysMet Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile 110 115 120 Ile ArgArg Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn 125 130 135 LysThr Cys Pro Thr Asn Tyr Met Trp Asn Asn His Ile Cys Arg Cys 140 145 150Leu Ala Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly Asp Asp Ser 155 160165 170 Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu Leu Asp Glu175 180 185 Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro Ala SerCys 190 195 200 Gly Pro His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys ValCys Lys 205 210 215 Asn Lys Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg GluPhe Asp Glu 220 225 230 Asn Thr Cys Gln Cys Val Cys Lys Arg Thr Cys ProArg Asn Gln Pro 235 240 245 250 Leu Asn Pro Gly Lys Cys Ala Cys Glu CysThr Glu Ser Pro Gln Lys 255 260 265 Cys Leu Leu Lys Gly Lys Lys Phe HisHis Gln Thr Cys Ser Cys Tyr 270 275 280 Arg Arg Pro Cys Thr Asn Arg GlnLys Ala Cys Glu Pro Gly Phe Ser 285 290 295 Tyr Ser Glu Glu Val Cys ArgCys Val Pro Ser Tyr Trp Lys Arg Pro 300 305 310 Gln Met Ser 315 22 basepairs nucleic acid single linear DNA (genomic) 34 TGAGTGATTTGTAGCTGCTGTG22 22 base pairs nucleic acid single linear DNA (genomic) 35TATTGCAGCAACCCCCACATCT 22 4416 base pairs nucleic acid single linear DNA(genomic) 36 CCACGCGCAG CGGCCGGAGA TGCAGCGGGG CGCCGCGCTG TGCCTGCGACTGTGGCTCTG 60 CCTGGGACTC CTGGACGGCC TGGTGAGTGG CTACTCCATG ACCCCCCCGACCTTGAACAT 120 CACGGAGGAG TCACACGTCA TCGACACCGG TGACAGCCTG TCCATCTCCTGCAGGGGACA 180 GCACCCCCTC GAGTGGGCTT GGCCAGGAGC TCAGGAGGCG CCAGCCACCGGAGACAAGGA 240 CAGCGAGGAC ACGGGGGTGG TGCGAGACTG CGAGGGCACA GACGCCAGGCCCTACTGCAA 300 GGTGTTGCTG CTGCACGAGG TACATGCCAA CGACACAGGC AGCTACGTCTGCTACTACAA 360 GTACATCAAG GCACGCATCG AGGGCACCAC GGCCGCCAGC TCCTACGTGTTCGTGAGAGA 420 CTTTGAGCAG CCATTCATCA ACAAGCCTGA CACGCTCTTG GTCAACAGGAAGGACGCCAT 480 GTGGGTGCCC TGTCTGGTGT CCATCCCCGG CCTCAATGTC ACGCTGCGCTCGCAAAGCTC 540 GGTGCTGTGG CCAGACGGGC AGGAGGTGGT GTGGGATGAC CGGCGGGGCATGCTCGTGTC 600 CACGCCACTG CTGCACGATG CCCTGTACCT GCAGTGCGAG ACCACCTGGGGAGACCAGGA 660 CTTCCTTTCC AACCCCTTCC TGGTGCACAT CACAGGCAAC GAGCTCTATGACATCCAGCT 720 GTTGCCCAGG AAGTCGCTGG AGCTGCTGGT AGGGGAGAAG CTGGTCCTGAACTGCACCGT 780 GTGGGCTGAG TTTAACTCAG GTGTCACCTT TGACTGGGAC TACCCAGGGAAGCAGGCAGA 840 GCGGGGTAAG TGGGTGCCCG AGCGACGCTC CCAGCAGACC CACACAGAACTCTCCAGCAT 900 CCTGACCATC CACAACGTCA GCCAGCACGA CCTGGGCTCG TATGTGTGCAAGGCCAACAA 960 CGGCATCCAG CGATTTCGGG AGAGCACCGA GGTCATTGTG CATGAAAATCCCTTCATCAG 1020 CGTCGAGTGG CTCAAAGGAC CCATCCTGGA GGCCACGGCA GGAGACGAGCTGGTGAAGCT 1080 GCCCGTGAAG CTGGCAGCGT ACCCCCCGCC CGAGTTCCAG TGGTACAAGGATGGAAAGGC 1140 ACTGTCCGGG CGCCACAGTC CACATGCCCT GGTGCTCAAG GAGGTGACAGAGGCCAGCAC 1200 AGGCACCTAC ACCCTCGCCC TGTGGAACTC CGCTGCTGGC CTGAGGCGCAACATCAGCCT 1260 GGAGCTGGTG GTGAATGTGC CCCCCCAGAT ACATGAGAAG GAGGCCTCCTCCCCCAGCAT 1320 CTACTCGCGT CACAGCCGCC AGGCCCTCAC CTGCACGGCC TACGGGGTGCCCCTGCCTCT 1380 CAGCATCCAG TGGCACTGGC GGCCCTGGAC ACCCTGCAAG ATGTTTGCCCAGCGTAGTCT 1440 CCGGCGGCGG CAGCAGCAAG ACCTCATGCC ACAGTGCCGT GACTGGAGGGCGGTGACCAC 1500 GCAGGATGCC GTGAACCCCA TCGAGAGCCT GGACACCTGG ACCGAGTTTGTGGAGGGAAA 1560 GAATAAGACT GTGAGCAAGC TGGTGATCCA GAATGCCAAC GTGTCTGCCATGTACAAGTG 1620 TGTGGTCTCC AACAAGGTGG GCCAGGATGA GCGGCTCATC TACTTCTATGTGACCACCAT 1680 CCCCGACGGC TTCACCATCG AATCCAAGCC ATCCGAGGAG CTACTAGAGGGCCAGCCGGT 1740 GCTCCTGAGC TGCCAAGCCG ACAGCTACAA GTACGAGCAT CTGCGCTGGTACCGCCTCAA 1800 CCTGTCCACG CTGCACGATG CGCACGGGAA CCCGCTTCTG CTCGACTGCAAGAACGTGCA 1860 TCTGTTCGCC ACCCCTCTGG CCGCCAGCCT GGAGGAGGTG GCACCTGGGGCGCGCCACGC 1920 CACGCTCAGC CTGAGTATCC CCCGCGTCGC GCCCGAGCAC GAGGGCCACTATGTGTGCGA 1980 AGTGCAAGAC CGGCGCAGCC ATGACAAGCA CTGCCACAAG AAGTACCTGTCGGTGCAGGC 2040 CCTGGAAGCC CCTCGGCTCA CGCAGAACTT GACCGACCTC CTGGTGAACGTGAGCGACTC 2100 GCTGGAGATG CAGTGCTTGG TGGCCGGAGC GCACGCGCCC AGCATCGTGTGGTACAAAGA 2160 CGAGAGGCTG CTGGAGGAAA AGTCTGGAGT CGACTTGGCG GACTCCAACCAGAAGCTGAG 2220 CATCCAGCGC GTGCGCGAGG AGGATGCGGG ACGCTATCTG TGCAGCGTGTGCAACGCCAA 2280 GGGCTGCGTC AACTCCTCCG CCAGCGTGGC CGTGGAAGGC TCCGAGGATAAGGGCAGCAT 2340 GGAGATCGTG ATCCTTGTCG GTACCGGCGT CATCGCTGTC TTCTTCTGGGTCCTCCTCCT 2400 CCTCATCTTC TGTAACATGA GGAGGCCGGC CCACGCAGAC ATCAAGACGGGCTACCTGTC 2460 CATCATCATG GACCCCGGGG AGGTGCCTCT GGAGGAGCAA TGCGAATACCTGTCCTACGA 2520 TGCCAGCCAG TGGGAATTCC CCCGAGAGCG GCTGCACCTG GGGAGAGTGCTCGGCTACGG 2580 CGCCTTCGGG AAGGTGGTGG AAGCCTCCGC TTTCGGCATC CACAAGGGCAGCAGCTGTGA 2640 CACCGTGGCC GTGAAAATGC TGAAAGAGGG CGCCACGGCC AGCGAGCACCGCGCGCTGAT 2700 GTCGGAGCTC AAGATCCTCA TTCACATCGG CAACCACCTC AACGTGGTCAACCTCCTCGG 2760 GGCGTGCACC AAGCCGCAGG GCCCCCTCAT GGTGATCGTG GAGTTCTGCAAGTACGGCAA 2820 CCTCTCCAAC TTCCTGCGCG CCAAGCGGGA CGCCTTCAGC CCCTGCGCGGAGAAGTCTCC 2880 CGAGCAGCGC GGACGCTTCC GCGCCATGGT GGAGCTCGCC AGGCTGGATCGGAGGCGGCC 2940 GGGGAGCAGC GACAGGGTCC TCTTCGCGCG GTTCTCGAAG ACCGAGGGCGGAGCGAGGCG 3000 GGCTTCTCCA GACCAAGAAG CTGAGGACCT GTGGCTGAGC CCGCTGACCATGGAAGATCT 3060 TGTCTGCTAC AGCTTCCAGG TGGCCAGAGG GATGGAGTTC CTGGCTTCCCGAAAGTGCAT 3120 CCACAGAGAC CTGGCTGCTC GGAACATTCT GCTGTCGGAA AGCGACGTGGTGAAGATCTG 3180 TGACTTTGGC CTTGCCCGGG ACATCTACAA AGACCCTGAC TACGTCCGCAAGGGCAGTGC 3240 CCGGCTGCCC CTGAAGTGGA TGGCCCCTGA AAGCATCTTC GACAAGGTGTACACCACGCA 3300 GAGTGACGTG TGGTCCTTTG GGGTGCTTCT CTGGGAGATC TTCTCTCTGGGGGCCTCCCC 3360 GTACCCTGGG GTGCAGATCA ATGAGGAGTT CTGCCAGCGG CTGAGAGACGGCACAAGGAT 3420 GAGGGCCCCG GAGCTGGCCA CTCCCGCCAT ACGCCGCATC ATGCTGAACTGCTGGTCCGG 3480 AGACCCCAAG GCGAGACCTG CATTCTCGGA GCTGGTGGAG ATCCTGGGGGACCTGCTCCA 3540 GGGCAGGGGC CTGCAAGAGG AAGAGGAGGT CTGCATGGCC CCGCGCAGCTCTCAGAGCTC 3600 AGAAGAGGGC AGCTTCTCGC AGGTGTCCAC CATGGCCCTA CACATCGCCCAGGCTGACGC 3660 TGAGGACAGC CCGCCAAGCC TGCAGCGCCA CAGCCTGGCC GCCAGGTATTACAACTGGGT 3720 GTCCTTTCCC GGGTGCCTGG CCAGAGGGGC TGAGACCCGT GGTTCCTCCAGGATGAAGAC 3780 ATTTGAGGAA TTCCCCATGA CCCCAACGAC CTACAAAGGC TCTGTGGACAACCAGACAGA 3840 CAGTGGGATG GTGCTGGCCT CGGAGGAGTT TGAGCAGATA GAGAGCAGGCATAGACAAGA 3900 AAGCGGCTTC AGGTAGCTGA AGCAGAGAGA GAGAAGGCAG CATACGTCAGCATTTTCTTC 3960 TCTGCACTTA TAAGAAAGAT CAAAGACTTT AAGACTTTCG CTATTTCTTCTACTGCTATC 4020 TACTACAAAC TTCAAAGAGG AACCAGGAGG ACAAGAGGAG CATGAAAGTGGACAAGGAGT 4080 GTGACCACTG AAGCACCACA GGGAAGGGGT TAGGCCTCCG GATGACTGCGGGCAGGCCTG 4140 GATAATATCC AGCCTCCCAC AAGAAGCTGG TGGAGCAGAG TGTTCCCTGACTCCTCCAAG 4200 GAAAGGGAGA CGCCCTTTCA TGGTCTGCTG AGTAACAGGT GCNTTCCCAGACACTGGCGT 4260 TACTGCTTGA CCAAAGAGCC CTCAAGCGGC CCTTATGCCA GCGTGACAGAGGGCTCACCT 4320 CTTGCCTTCT AGGTCACTTC TCACACAATG TCCCTTCAGC ACCTGACCCTGTGCCCGCCA 4380 GTTATTCCTT GGTAATATGA GTAATACATC AAAGAG 4416 4273 basepairs nucleic acid single linear DNA (genomic) 37 AAGCTTATCG ATTTCGAACCCGGGGGTACC GAATTCCTCG AGTCTAGAGG AGCATGCCTG 60 CAGGTCGACC GGGCTCGATCCCCTCGCGAG TTGGTTCAGC TGCTGCCTGA GGCTGGACGA 120 CCTCGCGGAG TTCTACCGGCAGTGCAAATC CGTCGGCATC CAGGAAACCA GCAGCGGCTA 180 TCCGCGCATC CATGCCCCCGAACTGCAGGA GTGGGGAGGC ACGATGGCCG CTTTGGTCCC 240 GGATCTTTGT GAAGGAACCTTACTTCTGTG GTGTGACATA ATTGGACAAA CTACCTACAG 300 AGATTTAAAG CTCTAAGGTAAATATAAAAT TTTTAAGTGT ATAATGTGTT AAACTACTGA 360 TTCTAATTGT TTGTGTATTTTAGATTCCAA CCTATGGAAC TGATGAATGG GAGCAGTGGT 420 GGAATGCCTT TAATGAGGAAAACCTGTTTT GCTCAGAAGA AATGCCATCT AGTGATGATG 480 AGGCTACTGC TGACTCTCAACATTCTACTC CTCCAAAAAA GAAGAGAAAG GTAGAAGACC 540 CCAAGGACTT TCCTTCAGAATTGCTAAGTT TTTTGAGTCA TGCTGTGTTT AGTAATAGAA 600 CTCTTGCTTG CTTTGCTATTTACACCACAA AGGAAAAAGC TGCACTGCTA TACAAGAAAA 660 TTATGGAAAA ATATTCTGTAACCTTTATAA GTAGGCATAA CAGTTATAAT CATAACATAC 720 TGTTTTTTCT TACTCCACACAGGCATAGAG TGTCTGCTAT TAATAACTAT GCTCAAAAAT 780 TGTGTACCTT TAGCTTTTTAATTTGTAAAG GGGTTAATAA GGAATATTTG ATGTATAGTG 840 CCTTGACTAG AGATCATAATCAGCCATACC ACATTTGTAG AGGTTTTACT TGCTTTAAAA 900 AACCTCCCAC ACCTCCCCCTGAACCTGAAA CATAAAATGA ATGCAATTGT TGTTGTTAAC 960 TTGTTTATTG CAGCTTATAATGGTTACAAA TAAAGCAATA GCATCACAAA TTTCACAAAT 1020 AAAGCATTTT TTTCACTGCATTCTAGTTGT GGTTTGTCCA AACTCATCAA TGTATCTTAT 1080 CATGTCTGGA TCTGCCGGTCTCCCTATAGT GAGTCGTATT AATTTCGATA AGCCAGGTTA 1140 ACCTGCATTA ATGAATCGGCCAACGCGCGG GGAGAGGCGG TTTGCGTATT GGGCGCTCTT 1200 CCGCTTCCTC GCTCACTGACTCGCTGCGCT CGGTCGTTCG GCTGCGGCGA GCGGTATCAG 1260 CTCACTCAAA GGCGGTAATACGGTTATCCA CAGAATCAGG GGATAACGCA GGAAAGAACA 1320 TGTGAGCAAA AGGCCAGCAAAAGGCCAGGA ACCGTAAAAA GGACGCGTTG CTGGCGTTTT 1380 TCCATAGGCT CCGCCCCCCTGACGAGCATC ACAAAAATCG ACGCTCAAGT CAGAGGTGGC 1440 GAAACCCGAC AGGACTATAAAGATACCAGG CGTTTCCCCC TGGAAGCTCC CTCGTGCGCT 1500 CTCCTGTTCC GACCCTGCCGCTTACCGGAT ACCTGTCCGC CTTTCTCCCT TCGGGAAGCG 1560 TGGCGCTTTC TCAATGCTCACGCTGTAGGT ATCTCAGTTC GGTGTAGGTC GTTCGCTCCA 1620 AGCTGGGCTG TGTGCACGAACCCCCCGTTC AGCCCGACCG CTGCGCCTTA TCCGGTAACT 1680 ATCGTCTTGA GTCCAACCCGGTAAGACACG ACTTATCGCC ACTGGCAGCA GCCACTGGTA 1740 ACAGGATTAG CAGAGCGAGGTATGTAGGCG GTGCTACAGA GTTCTTGAAG TGGTGGCCTA 1800 ACTACGGCTA CACTAGAAGGACAGTATTTG GTATCTGCGC TCTGCTGAAG CCAGTTACCT 1860 TCGGAAAAAG AGTTGGTAGCTCTTGATCCG GCAAACAAAC CACCGCTGGT AGCGGTGGTT 1920 TTTTTGTTTG CAAGCAGCAGATTACGCGCA GAAAAAAAGG ATCTCAAGAA GATCCTTTGA 1980 TCTTTTCTAC GGGGTCTGACGCTCAGTGGA ACGAAAACTC ACGTTAAGGG ATTTTGGTCA 2040 TGAGATTATC AAAAAGGATCTTCACCTAGA TCCTTTTAAA TTAAAAATGA AGTTTTAAAT 2100 CAATCTAAAG TATATATGAGTAAACTTGGT CTGACAGTTA CCAATGCTTA ATCAGTGAGG 2160 CACCTATCTC AGCGATCTGTCTATTTCGTT CATCCATAGT TGCCTGACTC CCCGTCGTGT 2220 AGATAACTAC GATACGGGAGGGCTTACCAT CTGGCCCCAG TGCTGCAATG ATACCGCGAG 2280 ACCCACGCTC ACCGGCTCCAGATTTATCAG CAATAAACCA GCCAGCCGGA AGGGCCGAGC 2340 GCAGAAGTGG TCCTGCAACTTTATCCGCCT CCATCCAGTC TATTAATTGT TGCCGGGAAG 2400 CTAGAGTAAG TAGTTCGCCAGTTAATAGTT TGCGCAACGT TGTTGCCATT GCTACAGGCA 2460 TCGTGGTGTC ACGCTCGTCGTTTGGTATGG CTTCATTCAG CTCCGGTTCC CAACGATCAA 2520 GGCGAGTTAC ATGATCCCCCATGTTGTGCA AAAAAGCGGT TAGCTCCTTC GGTCCTCCGA 2580 TCGTTGTCAG AAGTAAGTTGGCCGCAGTGT TATCACTCAT GGTTATGGCA GCACTGCATA 2640 ATTCTCTTAC TGTCATGCCATCCGTAAGAT GCTTTTCTGT GACTGGTGAG TACTCAACCA 2700 AGTCATTCTG AGAATAGTGTATGCGGCGAC CGAGTTGCTC TTGCCCGGCG TCAATACGGG 2760 ATAATACCGC GCCACATAGCAGAACTTTAA AAGTGCTCAT CATTGGAAAA CGTTCTTCGG 2820 GGCGAAAACT CTCAAGGATCTTACCGCTGT TGAGATCCAG TTCGATGTAA CCCACTCGTG 2880 CACCCAACTG ATCTTCAGCATCTTTTACTT TCACCAGCGT TTCTGGGTGA GCAAAAACAG 2940 GAAGGCAAAA TGCCGCAAAAAAGGGAATAA GGGCGACACG GAAATGTTGA ATACTCATAC 3000 TCTTCCTTTT TCAATATTATTGAAGCATTT ATCAGGGTTA TTGTCTCATG AGCGGATACA 3060 TATTTGAATG TATTTAGAAAAATAAACAAA TAGGGGTTCC GCGCACATTT CCCCGAAAAG 3120 TGCCACCTGA CGTCTAAGAAACCATTATTA TCATGACATT AACCTATAAA AATAGGCGTA 3180 TCACGAGGCC CTTTCGTCTCGCGCGTTTCG GTGATGACGG TGAAAACCTC TGACACATGC 3240 AGCTCCCGGA GACGGTCACAGCTTGTCTGT AAGCGGATGC CGGGAGCAGA CAAGCCCGTC 3300 AGGGCGCGTC AGCGGGTGTTGGCGGGTGTC GGGGCTGGCT TAACTATGCG GCATCAGAGC 3360 AGATTGTACT GAGAGTGCACCATATGGACA TATTGTCGTT AGAACGCGGC TACAATTAAT 3420 ACATAACCTT ATGTATCATACACATACGAT TTAGGTGACA CTATAGAACT CGAGCAGAGC 3480 TTCCAAATTG AGAGAGAGGCTTAATCAGAG ACAGAAACTG TTTGAGTCAA CTCAAGGATG 3540 GTTTGAGGGA CTGTTTAACAGATCCCCTTG GTTTACCACC TTGATATCTA CCATTATGGG 3600 ACCCCTCATT GTACTCCTAATGATTTTGCT CTTCGGACCC TGCATTCTTA ATCGATTAGT 3660 CCAATTTGTT AAAGACAGGATATCAGTGGT CCAGGCTCTA GTTTTGACTC AACAATATCA 3720 CCAGCTGAAG CCTATAGAGTACGAGCCATA GATAAAATAA AAGATTTTAT TTAGTCTCCA 3780 GAAAAAGGGG GGAATGAAAGACCCCACCTG TAGGTTTGGC AAGCTAGCTT AAGTAACGCC 3840 ATTTTGCAAG GCATGGAAAAATACATAACT GAGAATAGAG AAGTTCAGAT CAAGGTCAGG 3900 AACAGATGGA ACAGCTGAATATGGGCCAAA CAGGATATCT GTGGTAAGCA GTTCCTGCCC 3960 CGGCTCAGGG CCAAGAACAGATGGAACAGC TGAATATGGG CCAAACAGGA TATCTGTGGT 4020 AAGCAGTTCC TGCCCCGGCTCAGGGCCAAG AACAGATGGT CCCCAGATGC GGTCCAGCCC 4080 TCAGCAGTTT CTAGAGAACCATCAGATGTT TCCAGGGTGC CCCAAGGACC TGAAATGACC 4140 CTGTGCCTTA TTTGAACTAACCAATCAGTT CGCTTCTCGC TTCTGTTCGC GCGCTTCTGC 4200 TCCCCGAGCT CAATAAAAGAGCCCACAACC CCTCACTCGG GGCGCCAGTC CTCCGATTGA 4260 CTGAGTCGCC CGG 4273 216base pairs nucleic acid single linear DNA (genomic) 38 CAAGAAAGCGGCTTCAGCTG TAAAGGACCT GGCCAGAATG TGGCTGTGAC CAGGGCACAC 60 CCTGACTCCCAAGGGAGGCG GCGGCGGCCT GAGCGGGGGG CCCGAGGAGG CCAGGTGTTT 120 TACAACAGCGAGTATGGGGA GCTGTCGGAG CCAAGCGAGG AGGACCACTG CTCCCCGTCT 180 GCCCGCGTGACTTTCTTCAC AGACAACAGC TACTAA 216 17 amino acids amino acid single linearprotein 39 Glu Glu Thr Ile Lys Phe Ala Ala Ala His Tyr Asn Thr Glu IleLeu 1 5 10 15 Lys 1836 base pairs nucleic acid single linear DNA(genomic) CDS 168..1412 40 GCGGCCGCGT CGACGCAAAA GTTGCGAGCC GCCGAGTCCCGGGAGACGCT CGCCCAGGGG 60 GGTCCCCGGG AGGAAACCAC GGGACAGGGA CCAGGAGAGGACCTCAGCCT CACGCCCCAG 120 CCTGCGCCAG CCAACGGACC GGCCTCCCTG CTCCCGGTCCATCCACC ATG CAC TTG 176 Met His Leu 1 CTG TGC TTC TTG TCT CTG GCG TGTTCC CTG CTC GCC GCT GCG CTG ATC 224 Leu Cys Phe Leu Ser Leu Ala Cys SerLeu Leu Ala Ala Ala Leu Ile 5 10 15 CCC AGT CCG CGC GAG GCG CCC GCC ACCGTC GCC GCC TTC GAG TCG GGA 272 Pro Ser Pro Arg Glu Ala Pro Ala Thr ValAla Ala Phe Glu Ser Gly 20 25 30 35 CTG GGC TTC TCG GAA GCG GAG CCC GACGGG GGC GAG GTC AAG GCT TTT 320 Leu Gly Phe Ser Glu Ala Glu Pro Asp GlyGly Glu Val Lys Ala Phe 40 45 50 GAA GGC AAA GAC CTG GAG GAG CAG TTG CGGTCT GTG TCC AGC GTA GAT 368 Glu Gly Lys Asp Leu Glu Glu Gln Leu Arg SerVal Ser Ser Val Asp 55 60 65 GAG CTG ATG TCT GTC CTG TAC CCA GAC TAC TGGAAA ATG TAC AAG TGC 416 Glu Leu Met Ser Val Leu Tyr Pro Asp Tyr Trp LysMet Tyr Lys Cys 70 75 80 CAG CTG CGG AAA GGC GGC TGG CAG CAG CCC ACC CTCAAT ACC AGG ACA 464 Gln Leu Arg Lys Gly Gly Trp Gln Gln Pro Thr Leu AsnThr Arg Thr 85 90 95 GGG GAC AGT GTA AAA TTT GCT GCT GCA CAT TAT AAC ACAGAG ATC CTG 512 Gly Asp Ser Val Lys Phe Ala Ala Ala His Tyr Asn Thr GluIle Leu 100 105 110 115 AAA AGT ATT GAT AAT GAG TGG AGA AAG ACT CAA TGCATG CCA CGT GAG 560 Lys Ser Ile Asp Asn Glu Trp Arg Lys Thr Gln Cys MetPro Arg Glu 120 125 130 GTG TGT ATA GAT GTG GGG AAG GAG TTT GGA GCA GCCACA AAC ACC TTC 608 Val Cys Ile Asp Val Gly Lys Glu Phe Gly Ala Ala ThrAsn Thr Phe 135 140 145 TTT AAA CCT CCA TGT GTG TCC GTC TAC AGA TGT GGGGGT TGC TGC AAC 656 Phe Lys Pro Pro Cys Val Ser Val Tyr Arg Cys Gly GlyCys Cys Asn 150 155 160 AGG GAG GGG CTG CAG TGC ATG AAC ACC AGC ACA GGTTAC CTC AGC AAG 704 Arg Glu Gly Leu Gln Cys Met Asn Thr Ser Thr Gly TyrLeu Ser Lys 165 170 175 ACG TTG TTT GAA ATT ACA GTG CCT CTC TCA CAA GGCCCC AAA CCA GTC 752 Thr Leu Phe Glu Ile Thr Val Pro Leu Ser Gln Gly ProLys Pro Val 180 185 190 195 ACA ATC AGT TTT GCC AAT CAC ACT TCC TGC CGGTGC ATG TCT AAA CTG 800 Thr Ile Ser Phe Ala Asn His Thr Ser Cys Arg CysMet Ser Lys Leu 200 205 210 GAT GTT TAC AGA CAA GTT CAT TCA ATT ATT AGACGT TCT CTG CCA GCA 848 Asp Val Tyr Arg Gln Val His Ser Ile Ile Arg ArgSer Leu Pro Ala 215 220 225 ACA TTA CCA CAG TGT CAG GCA GCT AAC AAG ACATGT CCA ACA AAC TAT 896 Thr Leu Pro Gln Cys Gln Ala Ala Asn Lys Thr CysPro Thr Asn Tyr 230 235 240 GTG TGG AAT AAC TAC ATG TGC CGA TGC CTG GCTCAG CAG GAT TTT ATC 944 Val Trp Asn Asn Tyr Met Cys Arg Cys Leu Ala GlnGln Asp Phe Ile 245 250 255 TTT TAT TCA AAT GTT GAA GAT GAC TCA ACC AATGGA TTC CAT GAT GTC 992 Phe Tyr Ser Asn Val Glu Asp Asp Ser Thr Asn GlyPhe His Asp Val 260 265 270 275 TGT GGA CCC AAC AAG GAG CTG GAT GAA GACACC TGT CAG TGT GTC TGC 1040 Cys Gly Pro Asn Lys Glu Leu Asp Glu Asp ThrCys Gln Cys Val Cys 280 285 290 AAG GGG GGG CTT CGG CCA TCT AGT TGT GGACCC CAC AAA GAA CTA GAT 1088 Lys Gly Gly Leu Arg Pro Ser Ser Cys Gly ProHis Lys Glu Leu Asp 295 300 305 AGA GAC TCA TGT CAG TGT GTC TGT AAA AACAAA CTT TTC CCT AAT TCA 1136 Arg Asp Ser Cys Gln Cys Val Cys Lys Asn LysLeu Phe Pro Asn Ser 310 315 320 TGT GGA GCC AAC AGG GAA TTT GAT GAG AATACA TGT CAG TGT GTA TGT 1184 Cys Gly Ala Asn Arg Glu Phe Asp Glu Asn ThrCys Gln Cys Val Cys 325 330 335 AAA AGA ACG TGT CCA AGA AAT CAG CCC CTGAAT CCT GGG AAA TGT GCC 1232 Lys Arg Thr Cys Pro Arg Asn Gln Pro Leu AsnPro Gly Lys Cys Ala 340 345 350 355 TGT GAA TGT ACA GAA AAC ACA CAG AAGTGC TTC CTT AAA GGG AAG AAG 1280 Cys Glu Cys Thr Glu Asn Thr Gln Lys CysPhe Leu Lys Gly Lys Lys 360 365 370 TTC CAC CAT CAA ACA TGC AGT TGT TACAGA AGA CCG TGT GCG AAT CGA 1328 Phe His His Gln Thr Cys Ser Cys Tyr ArgArg Pro Cys Ala Asn Arg 375 380 385 CTG AAG CAT TGT GAT CCA GGA CTG TCCTTT AGT GAA GAA GTA TGC CGC 1376 Leu Lys His Cys Asp Pro Gly Leu Ser PheSer Glu Glu Val Cys Arg 390 395 400 TGT GTC CCA TCG TAT TGG AAA AGG CCACAT CTG AAC TAAGATCATA 1422 Cys Val Pro Ser Tyr Trp Lys Arg Pro His LeuAsn 405 410 415 CCAGTTTTCA GTCAGTCACA GTCATTTACT CTCTTGAAGA CTGTTGGAACAGCACTTAGC 1482 ACTGTCTATG CACAGAAAGA CTCTGTGGGA CCACATGGTA ACAGAGGCCCAAGTCTGTGT 1542 TTATTGAACC ATGTGGATTA CTGCGGGAGA GGACTGGCAC TCATGTGCAAAAAAAACCTC 1602 TTCAAAGACT GGTTTTCTGC CAGGGACCAG ACAGCTGAGG TTTTTCTCTTGTGATTTAAA 1662 AAAAGAATGA CTATATAATT TATTTCCACT AAAAATATTG TTCCTGCATTCATTTTTATA 1722 GCAATAACAA TTGGTAAAGC TCACTGTGAT CAGTATTTTT ATAACATGCAAAACTATGTT 1782 TAAAATAAAA TGAAAATTGT ATTATAAAAA AAAAAAAAAA AAAAAAAAAAGCTT 1836 415 amino acids amino acid linear protein 41 Met His Leu LeuCys Phe Leu Ser Leu Ala Cys Ser Leu Leu Ala Ala 1 5 10 15 Ala Leu IlePro Ser Pro Arg Glu Ala Pro Ala Thr Val Ala Ala Phe 20 25 30 Glu Ser GlyLeu Gly Phe Ser Glu Ala Glu Pro Asp Gly Gly Glu Val 35 40 45 Lys Ala PheGlu Gly Lys Asp Leu Glu Glu Gln Leu Arg Ser Val Ser 50 55 60 Ser Val AspGlu Leu Met Ser Val Leu Tyr Pro Asp Tyr Trp Lys Met 65 70 75 80 Tyr LysCys Gln Leu Arg Lys Gly Gly Trp Gln Gln Pro Thr Leu Asn 85 90 95 Thr ArgThr Gly Asp Ser Val Lys Phe Ala Ala Ala His Tyr Asn Thr 100 105 110 GluIle Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys Thr Gln Cys Met 115 120 125Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe Gly Ala Ala Thr 130 135140 Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr Arg Cys Gly Gly 145150 155 160 Cys Cys Asn Arg Glu Gly Leu Gln Cys Met Asn Thr Ser Thr GlyTyr 165 170 175 Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu Ser GlnGly Pro 180 185 190 Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser CysArg Cys Met 195 200 205 Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser IleIle Arg Arg Ser 210 215 220 Leu Pro Ala Thr Leu Pro Gln Cys Gln Ala AlaAsn Lys Thr Cys Pro 225 230 235 240 Thr Asn Tyr Val Trp Asn Asn Tyr MetCys Arg Cys Leu Ala Gln Gln 245 250 255 Asp Phe Ile Phe Tyr Ser Asn ValGlu Asp Asp Ser Thr Asn Gly Phe 260 265 270 His Asp Val Cys Gly Pro AsnLys Glu Leu Asp Glu Asp Thr Cys Gln 275 280 285 Cys Val Cys Lys Gly GlyLeu Arg Pro Ser Ser Cys Gly Pro His Lys 290 295 300 Glu Leu Asp Arg AspSer Cys Gln Cys Val Cys Lys Asn Lys Leu Phe 305 310 315 320 Pro Asn SerCys Gly Ala Asn Arg Glu Phe Asp Glu Asn Thr Cys Gln 325 330 335 Cys ValCys Lys Arg Thr Cys Pro Arg Asn Gln Pro Leu Asn Pro Gly 340 345 350 LysCys Ala Cys Glu Cys Thr Glu Asn Thr Gln Lys Cys Phe Leu Lys 355 360 365Gly Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr Arg Arg Pro Cys 370 375380 Ala Asn Arg Leu Lys His Cys Asp Pro Gly Leu Ser Phe Ser Glu Glu 385390 395 400 Val Cys Arg Cys Val Pro Ser Tyr Trp Lys Arg Pro His Leu Asn405 410 415

What is claimed is:
 1. An antibody which binds to a peptide consistingof the amino acid sequence set forth in SEQ ID NO:
 39. 2. A compositioncomprising an antibody according to claim 1, in apharmaceutically-acceptable diluent, adjuvant, or carrier.
 3. Anantibody according to claim 1 that is a monoclonal antibody.
 4. Acomposition comprising an antibody according to claim 3 in apharmaceutically-acceptable diluent, adjuvant, or carrier.
 5. Anantibody according to claim 3, further comprising a label.
 6. Anantibody according to claim 1, further comprising a label.